Antibody/T-cell receptor chimeric constructs and uses thereof

ABSTRACT

The present application provides antibody-TCR chimeric constructs comprising an antibody moiety that specifically binds to a target antigen fused to a TCRM capable of recruiting at least one TCR-associated signaling module. Also provided are methods of making and using these constructs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/121,475, filed on Sep. 4, 2018, which is continuation of U.S. patentapplication Ser. No. 15/829,793, filed on Dec. 1, 2017, now U.S. Pat.No. 10,098,951, which is a continuation of International Application No.PCT/US2016/058305, filed on Oct. 21, 2016, which claims priority to U.S.Provisional Application No. 62/245,944, filed on Oct. 23, 2015, U.S.Provisional Application No. 62/304,918, filed on Mar. 7, 2016, U.S.Provisional Application No. 62/345,649, filed on Jun. 3, 2016, and U.S.Provisional Application No. 62/369,694, filed on Aug. 1, 2016, all ofwhich are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention pertains to antibody/T cell receptor chimeric constructsand uses thereof including treating and diagnosing diseases.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 750042000303SEQLIST.TXT,date recorded: Oct. 14, 2019, size: 104 KB).

BACKGROUND OF THE INVENTION

T-cell mediated immunity is an adaptive process of developing antigen(Ag)—specific T lymphocytes to eliminate viruses, bacterial, parasiticinfections or malignant cells. It can also involve aberrant recognitionof self-antigen, leading to autoimmune inflammatory diseases. The Agspecificity of T lymphocytes is based on recognition through the T CellReceptor (TCR) of unique antigenic peptides presented by MajorHistocompatibility Complex (MHC) molecules on Ag-presenting cells (APC)(Broere, et al., Principles of Immunopharmacology, 2011). Each Tlymphocyte expresses a unique TCR on the cell surface as the result ofdevelopmental selection upon maturation in the thymus. The TCR occurs intwo forms as either an αβ heterodimer or as a γδ heterodimer. T cellsexpress either the αβ form or the γδ form TCR on the cell surface. Thefour chains, α/β/γ/δ, all have a characteristic extracellular structureconsisting of a highly polymorphic “immunoglobulin variable region”-likeN-terminal domain and an “immunoglobulin constant region”-like seconddomain. Each of these domains has a characteristic intra-domaindisulfide bridge. The constant region is proximal to the cell membrane,followed by a connecting peptide, a transmembrane region and a shortcytoplasmic tail. The covalent linkage between the 2 chains of theheterodimeric TCR is formed by the cysteine residue located within theshort connecting peptide sequence bridging the extracellular constantdomain and the transmembrane region which forms a disulfide bond withthe paired TCR chain cysteine residue at the corresponding position (TheT cell Receptor Factsbook, 2001).

The αβ and γδ TCRs are associated with the non-polymorphicmembrane-bound CD3 proteins to form the functional octameric TCR-CD3complex, consisting of the TCR heterodimer and three dimeric signalingmodules, CD3δ/ε, CD3γ/ε and CD3ζ/ζ or ζ/η. Ionizable residues in thetransmembrane domain of each subunit form a polar network ofinteractions that hold the complex together. For T cell activation, theTCR N-terminal variable regions recognize the peptide/MHC complexpresented on the surface of target cell, whereas the CD3 proteinsparticipate in signal transduction (Call et al., Cell. 111(7):967-79,2002; The T cell Receptor Factsbook, 2001).

αβ TCR, also called conventional TCR, is expressed on most lymphocytesand consists of the glycosylated polymorphic α and β chains. Differentαβ TCRs can discriminate among different peptides embedded in thesurfaces of MHC II (mostly expressed on APC cell surfaces) and MHC I(expressed on all nucleated cells) molecules, whose dimensions andshapes are relatively constant. The γδ TCR, though structurally similarto the αβ TCR, recognizes carbohydrate-, nucleotide-, orphosphor-carrying antigens in a fashion independent of MHC presentation(The T cell Receptor Factsbook, 2001; Girardi et al., J. Invest.Dermatol. 126(1):25-31, 2006; Hayes et al., Immunity. 16(6):827-38,2002).

Cell surface proteins constitute only a small fraction of the cellularproteins and most of these proteins are not tumor-specific. In contrast,mutated or oncogenic tumor-associated proteins are typicallyintracellularly located, nuclear, cytoplasmic or secretory. Mostintracellular proteins are exposed on the cell surface as part of anormal process of protein catabolism and presentation by MHC molecules.Intracellular proteins are usually degraded by the proteasome orendo/lysosomes, and the resulting specific peptide fragments bind to MHCclass I/II molecules. These peptide/MHC complexes are displayed at thecell surface where they provide targets for T cell recognition viapeptide/MHC TCR interaction (Scheinberg et al., Oncotarget. 4(5):647-8,2013; Cheever et al., Clin. Cancer Res. 15(17):5323-37, 2009).

In the past two decades, fundamental advances in immunology and tumorbiology, combined with the identification of a large number of tumorantigens, have led to significant progress in the field of cell-basedimmunotherapy. T cell therapy occupies a large space in the field ofcell-based immunotherapy, with the goal of treating cancer bytransferring autologous and ex vivo expanded T cells to patients, andhas resulted in some notable antitumor responses (Blattman et al.,Science. 305(5681):200-5, 2004). For example, the administration ofnaturally occurring tumor infiltrating lymphocytes (TILs) expanded exvivo mediated an objective response rate ranging from 50-70% in melanomapatients, including bulky invasive tumors at multiple sites involvingliver, lung, soft tissue and brain (Rosenberg et al., Nat. Rev. Cancer.8(4):299-308, 2008; Dudley M E et al., J. Clin. Oncol. 23(10):2346-57,2005).

A major limitation to the widespread application of TIL therapy is thedifficulty in generating human T cells with antitumor potential. As analternative approach, exogenous high-affinity TCRs can be introducedinto normal autologous T cells of the patients through T cellengineering. The adoptive transfer of these cells into lympho-depletedpatients has been shown to mediate cancer regression in cancers such asmelanoma, colorectal carcinoma, and synovial sarcoma (Kunert R et al.,Front. Immunol. 4:363, 2013). A recent phase I clinical trial using antiNY-ESO-1 TCRs against synovial sarcoma reported an overall response rateof 66% and complete response was achieved in one of the patientsreceiving the T cell therapy (Robbins P F et al., Clin. Cancer Res.21(5):1019-27, 2015).

One of the advantages of TCR-engineered T cell therapy is that it cantarget the entire array of potential intracellular tumor-specificproteins, which are processed and delivered to the cell surface throughMHC presentation. Furthermore, the TCR is highly sensitive and can beactivated by just a few antigenic peptide/MHC molecules, which in turncan trigger a cytolytic T cell response, including cytokine secretion, Tcell proliferation and cytolysis of defined target cells. Therefore,compared with antibody or small molecule therapies, TCR-engineered Tcells are particularly valuable for their ability to kill target cellswith very few copies of target intracellular antigens (Kunert R et al.,Front. Immunol. 4:363, 2013).

However, unlike therapeutic antibodies, which are mostly discoveredthrough hybridoma or display technologies, identification oftarget-specific TCRs requires the establishment of target peptide/MHCspecific TCR clones from patient T cells and screening for the right α-βchain combination that has the optimal target antigen-binding affinity.Very often, phage/yeast display is employed after cloning of the TCRfrom patient T cells to further enhance the target binding affinity ofthe TCR. The whole process requires expertise in many areas and istime-consuming (Kobayashi E et al., Oncoimmunology. 3(1):e27258, 2014).The difficulties in the TCR discovery process have largely impeded thewidespread application of TCR-engineered T cell therapy. It has alsobeen hampered by treatment-related toxicity, in particularly with TCRsagainst antigens that are over-expressed on tumor cells but alsoexpressed on healthy cells, or with TCRs recognizing off-targetpeptide/MHC complexes (Rosenberg S A et al., Science. 348(6230):62-8,2015).

A different approach has been developed in recent years to engage Tcells for targeted cancer immunotherapy. This new approach is calledChimeric Antigen Receptor T cell Therapy (CAR-T). It merges theexquisite targeting specificity of monoclonal antibodies with the potentcytotoxicity and long-term persistence provided by cytotoxic T cells. ACAR is composed of an extracellular domain that recognizes a cellsurface antigen, a transmembrane region, and an intracellular signalingdomain. The extracellular domain consists of the antigen-bindingvariable regions from the heavy and light chains of a monoclonalantibody that are fused into a single-chain variable fragment (scFv).The intracellular signaling domain contains an immunoreceptortyrosine-based activation motif (ITAM), such as those from CD3ζ or FcRγ,and one or more costimulatory signaling domains, such as those fromCD28, 4-1BB or OX40 (Barrett D M et al., Annu. Rev. Med. 65:333-47,2014; Davila M L et al., Oncoimmunology. 1(9):1577-1583, 2012). Bindingof target antigens by CARs grafted onto a T cell surface can trigger Tcell effector functions independent of TCR-peptide/MHC complexinteraction. Thus, T cells equipped with CARs can be redirected toattack a broad variety of cells, including those that do not match theMHC type of the TCRs on the T cells but express the target cell-surfaceantigens. This approach overcomes the constraints of MHC-restricted TCRrecognition and avoids tumor escape through impairments in antigenpresentation or MHC molecule expression. Clinical trials have shownclinically significant antitumor activity of CAR-T therapy inneuroblastoma (Louis C U et al., Blood. 118(23):6050-6056, 2011), B-ALL(Maude, S L, et al., New England Journal of Medicine 371:16:1507-1517,2014), CLL (Brentjens, R J, et al. Blood 118:18:4817-4828, 2011), and Bcell lymphoma (Kochenderfer, J N, et al. Blood 116:20:4099-4102, 2010).In one study, a 90% complete remission rate in 30 patients with B-ALLtreated with CD19-CAR T therapy was reported (Maude, S L, et al.,supra).

Most, if not all, CARs studied so far have been directed to tumorantigens with high cell surface expression. To target low-copy numbercell-surface tumor antigens and intracellular tumor antigens, whichrepresent 95% of all known tumor-specific antigens, there is a need todevelop more potent and effective engineered cell therapies (Cheever, etal., Clin. Cancer Res. 15(17):5323-37, 2009).

Several attempts have been made to engineer chimeric receptor moleculeshaving antibody specificity with T cell receptor effector functions.See, for example, Kuwana, Y, et al., Biochem. Biophys. Res. Commun.149(3):960-968, 1987; Gross, G, et al., Proc. Natl. Acad. Sci. USA.86:10024-10028, 1989; Gross, G & Eshhar, Z, FASEB J. 6(15):3370-3378,1992; U.S. Pat. No. 7,741,465. To this date, none of these chimericreceptors have been adopted for clinical use, and novel designs forantibody-TCR chimeric receptors with improved expression andfunctionality in human T cells are needed.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present application in one aspect provides a construct (such as anisolated construct) comprising an antibody moiety (such as a Fab-likeantigen-binding module) fused to a T cell receptor module (saidconstruct also referred to herein as an “antibody-TCR chimericmolecule,” or “abTCR”). In some embodiments, the abTCR comprises aFab-like antigen-binding module that specifically binds to a targetantigen and a T cell receptor module (TCRM) capable of recruiting atleast one TCR-associated signaling module. In some embodiments, thetarget antigen is a complex comprising a peptide and an MHC protein(such as an MHC class I protein or an MHC class II protein). In someembodiments, the target antigen is a cell-surface antigen.

In some embodiments, there is provided an abTCR (such as an isolatedabTCR) that specifically binds to a target antigen, wherein the abTCRcomprises: a) a first polypeptide chain comprising a firstantigen-binding domain comprising V_(H) and C_(H)1 antibody domains anda first T cell receptor domain (TCRD) comprising a first transmembranedomain of a first TCR subunit; and b) a second polypeptide chaincomprising a second antigen-binding domain comprising V_(L) and C_(L)antibody domains and a second TCRD comprising a second transmembranedomain of a second TCR subunit, wherein the V_(H) and C_(H)1 domains ofthe first antigen-binding domain and the V_(L) and C_(L) domains of thesecond antigen-binding domain form a Fab-like antigen-binding modulethat specifically binds to the target antigen, and wherein the firstTCRD and the second TCRD form a T cell receptor module (TCRM) that iscapable of recruiting at least one TCR-associated signaling module. Insome embodiments, the first polypeptide chain and the second polypeptidechain are linked via one or more disulfide bonds. In some embodiments,the Fab-like antigen-binding module comprises a disulfide bond between aresidue in the C_(H)1 domain in the first polypeptide chain and aresidue in the C_(L) domain in the second polypeptide chain. In someembodiments, the first polypeptide chain further comprises a firstpeptide linker between the first antigen-binding domain and the firstTCRD. In some embodiments, the second polypeptide chain furthercomprises a second peptide linker between the second antigen-bindingdomain and the second TCRD. In some embodiments, the first peptidelinker and/or the second peptide linker are, individually, from about 5to about 50 amino acids in length. In some embodiments, the targetantigen is a cell surface antigen. In some embodiments, the cell surfaceantigen is selected from the group consisting of protein, carbohydrate,and lipid. In some embodiments, the cell surface antigen is CD19, ROR1,ROR2, BCMA, GPRC5D, or FCRL5. In some embodiments, the target antigen isa complex comprising a peptide and a major histocompatibility complex(MHC) protein.

In some embodiments, there is provided an abTCR that specifically bindsto a target antigen, comprising: a) a first polypeptide chain comprisinga first antigen-binding domain comprising a V_(H) antibody domain and afirst TCRD comprising a first transmembrane domain of a first TCRsubunit; and b) a second polypeptide chain comprising a secondantigen-binding domain comprising a V_(L) antibody domains and a secondTCRD comprising a second transmembrane domain of a second TCR subunit,wherein the V_(H) domain of the first antigen-binding domain and theV_(L) domain of the second antigen-binding domain form anantigen-binding module that specifically binds to the target antigen,wherein the first TCRD and the second TCRD form a T cell receptor module(TCRM) that is capable of recruiting at least one TCR-associatedsignaling module, and wherein the target antigen is a complex comprisinga peptide and an MHC protein. In some embodiments, the first polypeptidechain further comprises a first peptide linker between the firstantigen-binding domain and the first TCRD and the second polypeptidechain further comprises a second peptide linker between the secondantigen-binding domain and the second TCRD. In some embodiments, thefirst and/or second peptide linkers comprise, individually, a constantdomain or fragment thereof from an immunoglobulin or T cell receptorsubunit. In some embodiments, the first and/or second peptide linkerscomprise, individually, a CH1, CH2, CH3, CH4 or CL antibody domain, or afragment thereof. In some embodiments, the first and/or second peptidelinkers comprise, individually, a Cα, Cβ, Cγ, or Cδ TCR domain, or afragment thereof.

In some embodiments, according to any of the abTCRs (such as isolatedabTCRs) described above, the first TCRD further comprises a firstconnecting peptide or fragment thereof of a TCR subunit N-terminal tothe first transmembrane domain, the second TCRD further comprises asecond connecting peptide or fragment thereof of a TCR subunitN-terminal to the second transmembrane domain. In some embodiments, theTCRM comprises a disulfide bond between a residue in the firstconnecting peptide and a residue in the second connecting peptide. Insome embodiments, the first TCRD further comprises a first TCRintracellular domain comprising a TCR intracellular sequence C-terminalto the first transmembrane domain. In some embodiments, the second TCRDfurther comprises a second TCR intracellular domain comprising a TCRintracellular sequence C-terminal to the second transmembrane domain. Insome embodiments, the abTCR binds to the target antigen with anequilibrium dissociation constant (K_(d)) from about 0.1 pM to about 500nM. In some embodiments, the TCR-associated signaling module is selectedfrom the group consisting of CD3δε, CD3γεE, and ζζ.

In some embodiments, according to any of the abTCRs (such as isolatedabTCRs) described above, the first polypeptide chain further comprises afirst accessory intracellular domain comprising a co-stimulatoryintracellular signaling sequence C-terminal to the first transmembranedomain. In some embodiments, the second polypeptide chain furthercomprises a second accessory intracellular domain comprising aco-stimulatory intracellular signaling sequence C-terminal to the secondtransmembrane domain. In some embodiments, the first polypeptide chainfurther comprises a first signaling peptide N-terminal to the firstantigen-binding domain. In some embodiments, the second polypeptidechain further comprises a second signaling peptide N-terminal to thesecond antigen-binding domain.

In some embodiments, according to any of the abTCRs (such as isolatedabTCRs) described above where the target antigen is a complex comprisinga peptide and a major histocompatibility complex (MHC) protein, thepeptide is derived from a protein selected from the group consisting ofWT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, and PSA.

In some embodiments, according to any of the abTCRs (such as isolatedabTCRs) described above, a) the first TCR subunit is a TCR α chain, andthe second TCR subunit is a TCR β chain; b) the first TCR subunit is aTCR β chain, and the second TCR subunit is a TCR α chain; c) the firstTCR subunit is a TCR γ chain, and the second TCR subunit is a TCR δchain; or d) the first TCR subunit is a TCR δ chain, and the second TCRsubunit is a TCR γ chain.

In some embodiments, according to any of the abTCRs (such as isolatedabTCRs) described above, there is provided a nucleic acid encoding thefirst and second polypeptide chains of the abTCR.

In some embodiments, according to any of the abTCRs (such as isolatedabTCRs) described above, there is provided complex comprising the abTCRand at least one TCR-associated signaling module selected from the groupconsisting of CD3δε, CD3γε, and ζζ. In some embodiments, the complex isan octamer comprising the abTCR and CD3δε, CD3γε, and ζζ.

In some embodiments, according to any of the abTCRs (such as isolatedabTCRs) described above, there is provided an effector cell presentingon its surface the abTCR. In some embodiments, the effector cellcomprises a nucleic acid encoding the abTCR. In some embodiments, theeffector cell does not express the first TCR subunit and/or the secondTCR subunit. For example, in some embodiments, a) the first TCR subunitis TCRγ and the second TCR subunit is TCRδ; or b) the first TCR subunitis TCRδ and the second TCR subunit is TCRγ; and the effector cell is anαβ T cell. In some embodiments, a) the first TCR subunit is TCRγ and thesecond TCR subunit is TCRδ; or b) the first TCR subunit is TCRδ and thesecond TCR subunit is TCRγ; and the effector cell is an αβ T cell. Insome embodiments, the effector cell is modified to block or decrease theexpression of a first endogenous TCR subunit and/or a second endogenousTCR subunit. For example, in some embodiments, the first TCR subunit isTCRα and the second TCR subunit is TCRβ; or b) the first TCR subunit isTCRβ and the second TCR subunit is TCRα; and the effector cell is an αβT cell modified to block or decrease the expression of TCRα and/or TCRβ.In some embodiments, a) the first TCR subunit is TCRγ and second TCRsubunit is TCRδ; or b) the first TCR subunit is TCRδ and the second TCRsubunit is TCRγ; and the effector cell is a γδ T cell modified to blockor decrease the expression of TCRγ and/or TCRδ.

In some embodiments, according to any of the abTCRs (such as isolatedabTCRs) described above, there is provided an effector cell presentingon its surface the abTCR, wherein the effector cell is a T cell. In someembodiments, the T cell is selected from the group consisting of acytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, according to any of the abTCRs (such as isolatedabTCRs) described above, there is provided an effector cell presentingon its surface the abTCR, wherein the effector cell comprises a) a firstvector comprising a first nucleic acid sequence encoding the firstpolypeptide chain of the abTCR under the control of a first promoter andb) a second vector comprising a second nucleic acid sequence encodingthe second polypeptide chain of the abTCR under the control of a secondpromoter.

In some embodiments, according to any of the abTCRs (such as isolatedabTCRs) described above, there is provided an effector cell presentingon its surface the abTCR, wherein the effector cell comprises a vectorcomprising a) a first nucleic acid sequence encoding the firstpolypeptide chain of the abTCR under the control of a first promoter;and b) a second nucleic acid sequence encoding the second polypeptidechain of the abTCR under the control of a second promoter.

In some embodiments, according to any of the abTCRs (such as isolatedabTCRs) described above, there is provided an effector cell presentingon its surface the abTCR, wherein the effector cell comprises a vectorcomprising a) a first nucleic acid sequence encoding the firstpolypeptide chain of the abTCR and a second nucleic acid sequenceencoding the second polypeptide chain of the abTCR, wherein the firstand second nucleic acid sequences are under the control of a singlepromoter.

In some embodiments, according to any of the abTCRs (such as isolatedabTCRs) described above, there is provided an effector cell presentingon its surface the abTCR, wherein the expression of the firstpolypeptide chain of the abTCR is more than two-fold different than theexpression of the second polypeptide chain of the abTCR.

In some embodiments, there is provided a method of killing a target cellpresenting a target antigen, comprising contacting the target cell withan effector cell expressing an abTCR according to any of the abTCRs(such as isolated abTCRs) described above, wherein the abTCRspecifically binds to the target antigen.

In some embodiments, there is provided a method of killing a target cellpresenting a target antigen, comprising contacting the target cell withan effector αβ T cell comprising an abTCR that specifically binds to thetarget antigen comprising: a) a first polypeptide chain comprising afirst antigen-binding domain comprising a V_(H) antibody domain and afirst TCRD comprising a first transmembrane domain of a first TCRsubunit; and b) a second polypeptide chain comprising a secondantigen-binding domain comprising a V_(L) antibody domains and a secondTCRD comprising a second transmembrane domain of a second TCR subunit,wherein the V_(H) domain of the first antigen-binding domain and theV_(L) domain of the second antigen-binding domain form anantigen-binding module that specifically binds to the target antigen,wherein the first TCRD and the second TCRD form a T cell receptor module(TCRM) that is capable of recruiting at least one TCR-associatedsignaling module, and wherein the first TCR subunit is TCRγ and thesecond TCR subunit is TCRδ, or the first TCR subunit is TCR and thesecond TCR subunit is TCRγ. In some embodiments, the first polypeptidechain further comprises a first peptide linker between the firstantigen-binding domain and the first TCRD and the second polypeptidechain further comprises a second peptide linker between the secondantigen-binding domain and the second TCRD. In some embodiments, thefirst and/or second peptide linkers comprise, individually, a constantdomain or fragment thereof from an immunoglobulin or T cell receptorsubunit. In some embodiments, the first and/or second peptide linkerscomprise, individually, a CH1, CH2, CH3, CH4 or CL antibody domain, or afragment thereof. In some embodiments, the first and/or second peptidelinkers comprise, individually, a Cα, Cβ, Cγ, or Cδ TCR domain, or afragment thereof.

In some embodiments, according to any of the methods of killing a targetcell described aboved, the contacting is in vivo. In some embodiments,the contacting is in vitro.

In some embodiments, there is provided a pharmaceutical compositioncomprising an abTCR according to any of the abTCRs (such as isolatedabTCRs) described above and a pharmaceutically acceptable carrier. Insome embodiments, there is provided a pharmaceutical compositioncomprising a nucleic acid encoding an abTCR according to any of theembodiments described above and a pharmaceutically acceptable carrier.In some embodiments, there is provided a pharmaceutical compositioncomprising an effector cell expressing an abTCR according to any of theabTCRs (such as isolated abTCRs) described above and a pharmaceuticallyacceptable carrier.

In some embodiments, there is provided a method of treating a targetantigen-associated disease in an individual in need thereof comprisingadministering to the individual an effective amount of a pharmaceuticalcomposition comprising an effector cell expressing an abTCR according toany of the abTCRs (such as isolated abTCRs) described above.

In some embodiments, there is provided a method of treating a targetantigen-associated disease in an individual in need thereof comprisingadministering to the individual an effective amount of a compositioncomprising an effector αβ T cell comprising an abTCR that specificallybinds to the target antigen comprising: a) a first polypeptide chaincomprising a first antigen-binding domain comprising a V_(H) antibodydomain and a first TCRD comprising a first transmembrane domain of afirst TCR subunit; and b) a second polypeptide chain comprising a secondantigen-binding domain comprising a V_(L) antibody domains and a secondTCRD comprising a second transmembrane domain of a second TCR subunit,wherein the V_(H) domain of the first antigen-binding domain and theV_(L) domain of the second antigen-binding domain form anantigen-binding module that specifically binds to the target antigen,wherein the first TCRD and the second TCRD form a T cell receptor module(TCRM) that is capable of recruiting at least one TCR-associatedsignaling module, and wherein the first TCR subunit is TCRγ and thesecond TCR subunit is TCRδ, or the first TCR subunit is TCR and thesecond TCR subunit is TCRγ. In some embodiments, the wherein the firstpolypeptide chain further comprises a first peptide linker between thefirst antigen-binding domain and the first TCRD and the secondpolypeptide chain further comprises a second peptide linker between thesecond antigen-binding domain and the second TCRD. In some embodiments,the first and/or second peptide linkers comprise, individually, aconstant domain or fragment thereof from an immunoglobulin or T cellreceptor subunit. In some embodiments, the first and/or second peptidelinkers comprise, individually, a CH1, CH2, CH3, CH4 or CL antibodydomain, or a fragment thereof. In some embodiments, the first and/orsecond peptide linkers comprise, individually, a Cα, Cβ, Cγ, or Cδ TCRdomain, or a fragment thereof.

In some embodiments, according to any of the methods of treating atarget antigen-associated disease described above, the targetantigen-associated disease is cancer. In some embodiments, the cancer isselected from the group consisting of adrenocortical carcinoma, bladdercancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectalcancers, esophageal cancer, glioblastoma, glioma, hepatocellularcarcinoma, head and neck cancer, kidney cancer, lymphoma, leukemia, lungcancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer,pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostatecancer, sarcoma, stomach cancer, uterine cancer and thyroid cancer. Insome embodiments, the target antigen-associated disease is viralinfection. In some embodiments, the viral infection is caused by a virusselected from the group consisting of Cytomegalovirus (CMV),Epstein-Barr Virus (EBV), Hepatitis B Virus (HBV), Kaposi's Sarcomaassociated herpesvirus (KSHV), Human papillomavirus (HPV), Molluscumcontagiosum virus (MCV), Human T cell leukemia virus 1 (HTLV-1), HIV(Human immunodeficiency virus), and Hepatitis C Virus (HCV).

In some embodiments, there is provided a method of treating a targetantigen-associated disease in an individual in need thereof comprisingadministering to the individual an effective amount of a pharmaceuticalcomposition comprising a nucleic acid encoding an abTCR according to anyof the abTCRs (such as isolated abTCRs) described above.

In some embodiments, there is provided a method of enriching aheterogeneous cell population for an effector cell expressing an abTCRaccording to any of the abTCRs (such as isolated abTCRs) describedabove, wherein the method comprises a) contacting the heterogeneous cellpopulation with a ligand comprising the target antigen or one or moreepitopes contained therein to form complexes of the effector cell boundto the ligand; and b) separating the complexes from the heterogeneouscell population, thereby generating a cell population enriched for theeffector cell.

In some embodiments, there is provided a nucleic acid library comprisingsequences encoding a plurality of abTCRs according to any of the abTCRs(such as isolated abTCRs) described above.

In some embodiments, there is provided a method of screening a nucleicacid library according to any of the embodiments described above forsequences encoding abTCRs specific for a target antigen, comprising: a)introducing the nucleic acid library into a plurality of cells, suchthat the abTCRs are expressed on the surface of the plurality of cells;b) incubating the plurality of cells with a ligand comprising the targetantigen or one or more epitopes contained therein; c) collecting cellsbound to the ligand; and d) isolating sequences encoding the abTCRs fromcells collected in step c), thereby identifying abTCRs specific for thetarget antigen.

Also provided are methods of making any of the constructs describedherein, articles of manufacture, and kits that are suitable for themethods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic representation of the various abTCR constructdesigns (abTCR-3, abTCR-4, abTCR-5, and abTCR-6).

FIG. 1B shows contemplated variations of the abTCR construct designs.

FIG. 2 shows a conventional model for the assembly of the TCR-CD3complex.

FIG. 3 shows Western blot analysis of lysates from J.RT3-T3.5 or Jurkatcells individually transduced with abTCR-3, -4, -5, -6, or -6MDconstructs having an anti-AFP158/HLA*02:01 binding moiety, stained withanti-FLAG (TCRα- and TCRγ-derived chimeric subunits) or anti-HAantibodies (TCRβ- and TCRδ-derived chimeric subunits).

FIG. 4A shows flow cytometry analysis of surface CD3ε expression inJ.RT3-T3.5 cells individually transduced with abTCR-3, -4, -5, -6, or-6MD constructs having an anti-AFP158/HLA-A*02:01 binding moiety; cellswere stained with anti-CD3ε antibody.

FIG. 4B shows flow cytometry analysis of surface AFP158/HLA-A*02:01tetramer binding in J.RT3-T3.5 cells individually transduced withabTCR-3, -4, -5, -6, or -6MD constructs having ananti-AFP158/HLA-A*02:01 binding moiety; cells were stained withphycoerythrin (PE)-labeled AFP158/HLA-A*02:01 tetramers.

FIG. 4C shows flow cytometry analysis of surface anti-idiotype antibodybinding in J.RT3-T3.5 cells individually transduced with abTCR-3, -4,-5, -6, or -6MD constructs having an anti-AFP158/HLA-A*02:01 bindingmoiety recognized by the antibody; cells were stained with anti-idiotypeantibody against the anti-AFP158/HLA-A*02:01 binding moiety of the abTCRconstructs.

FIG. 5A shows flow cytometry analysis of surface anti-TCRα/β antibodybinding in Jurkat cells individually transduced with abTCR-3, -4, -5,-6, or -6MD constructs having an anti-AFP158/HLA-A*02:01 binding moiety;cells were stained with anti-TCRα/β antibody.

FIG. 5B shows flow cytometry analysis of surface AFP158/HLA-A*02:01tetramer binding in Jurkat cells individually transduced with abTCR-3,-4, -5, -6, or -6MD constructs having an anti-AFP158/HLA-A*02:01 bindingmoiety; cells were stained with PE-labeled AFP158/HLA-A*02:01 tetramers.

FIG. 5C shows flow cytometry analysis of surface anti-idiotype antibodybinding in Jurkat cells individually transduced with abTCR-3, -4, -5,-6, or -6MD constructs having an anti-AFP158/HLA-A*02:01 binding moietyrecognized by the antibody; cells were stained with anti-idiotypeantibody against the anti-AFP158/HLA-A*02:01 binding moiety of the abTCRconstructs.

FIG. 6 shows flow cytometry analysis of the co-expression of CD3ε withabTCR chimeras in J.RT3-T3.5 cells individually transduced with abTCR-6or abTCR-6MD constructs having an anti-AFP158/HLA-A*02:01 bindingmoiety; cells were co-stained with anti-CD3ε antibody andAFP158/HLA-A*02:01 tetramers.

FIG. 7A shows flow cytometry analysis of abTCR-transduced peripheralblood lymphocytes; cells were transduced with an abTCR-6MD constructhaving an anti-AFP158/HLA-A*02:01 binding moiety and co-stained withanti-CD4 antibody, anti-CD8 antibody and AFP158/HLA-A*02:01 tetramers.The dotted box indicates the tetramer⁺ population gate for the cellsshown in the CD4/CD8 plot in FIG. 7B.

FIG. 7B shows flow cytometry analysis of CD4 and CD8 expression onperipheral blood lymphocytes that were either mock-transduced ortransduced with an abTCR-6MD construct having an anti-AFP158/HLA-A*02:01binding moiety and co-stained with anti-CD4 and anti-CD8 antibodies; CD4and CD8 expression are shown for ungated cells (top 2 panels) ortetramer⁺ gated cells (bottom panel).

FIG. 8 shows Western blot analysis of the association of exogenous abTCRchains with the CD3 complex; Digitonin lysates were made from primary Tcells that were either mock-transduced or transduced with abTCR-6MDhaving an anti-AFP158/HLA-A*02:01 binding moiety; lysates or anti-FLAGimmunoprecipitates were blotted with anti-FLAG, anti-CD3δ, anti-CD3ε,anti-CD3γ or anti-CD3ζ antibodies.

FIG. 9A shows transduction efficiency in primary T cells after they weretransduced with a CAR or an abTCR-6MD, both having the sameanti-AFP158/HLA-A*02:01 binding moiety variable domains; cells werestained with PE-labeled AFP158/HLA-A*02:01 tetramers.

FIG. 9B shows killing of cancer cell lines HepG2, SK-HEP-1 andSK-HEP-1-AFP-MG, mediated by T cells transduced with either a CAR or anabTCR-6MD construct, both having the same anti-AFP158/HLA-A*02:01binding moiety variable domains.

FIG. 10 shows flow cytometry analysis of the degranulation ofabTCR-transduced T cells after co-culturing with target cells; T cellswere transduced with either a CAR or an abTCR-6MD, both having the sameanti-AFP158/HLA-A*02:01 binding moiety variable domains. Staining of thetransduced cells with AFP158/HLA-A*02:01 tetramers, anti-CD8 antibody oranti-CD107a antibody after co-culturing with target cells HepG2,SK-HEP-1 and SK-HEP-1-AFP-MG are shown.

FIG. 11A shows the level of secretion of a panel of cytokines bymock-transduced T cells or T cells transduced with either a CAR or anabTCR-6MD, both having the same anti-AFP158/HLA-A*02:01 binding moietyvariable domains, after co-culture with HepG2 cells.

FIG. 11B shows the level of secretion of a panel of cytokines bymock-transduced T cells or T cells transduced with either a CAR or anabTCR-6MD, both having the same anti-AFP158/HLA-A*02:01 binding moietyvariable domains, co-cultured with either SK-HEP-1 or SK-HEP-1-AFP-MGcells.

FIGS. 12A-12H show flow cytometry analysis of transduced T cells forcytokine production with or without the presence of target cancer cells;T cells were transduced with either a CAR or an abTCR-6MD, both havingthe same anti-AFP158/HLA-A*02:01 binding moiety variable domains,co-cultured with either SK-HEP-1, SK-HEP-1-AFP-MG, or HepG2 cells; cellswere subsequently co-stained with PE-labeled AFP158/HLA-A*02:01tetramers, anti-CD4 antibody and one of anti-TNF-α antibody (12A and12B), anti-IFNγ antibody (12C and 12D), anti-IL-2 antibody (12E and12F), or anti-IL-6 antibody (12G and 12H). Populations shown were gatedon AFP158/HLA-A*02:01 tetramer⁺ cells.

FIG. 13 shows target-specific activation of cytokine expression in CD4+T cells transduced with an anti-AFP158 abTCR and incubated with cancercell lines positive or negative for AFP expression.

FIG. 14 shows the flow cytometry analysis of T cell exhaustion markersPD-1, LAG-3 and TIM-3 on CAR- or abTCR-transduced T cells, both havingthe same anti-AFP158/HLA-A*02:01 binding moiety variable domains, uponexposure to antigen-positive or -negative target cells.

FIG. 15 shows flow cytometry analysis of T cell differentiation markersCD28, CCR7 and granzyme B on CAR- or abTCR-transduced T cells, bothhaving the same anti-AFP158/HLA-A*02:01 binding moiety variable domains,upon exposure to antigen-positive or -negative target cells.

FIGS. 16A-16C show the characterization of T cells transduced witheither an anti-AFP158/HLA-A*02:01 abTCR-6MD or ananti-AFP158/HLA-A*02:01 abTCR-7, both having the sameanti-AFP158/HLA-A*02:01 binding moiety variable domains. FIG. 16A showscell growth of the transduced T cells. FIG. 16B shows Western blotanalysis for expression of the abTCR-6MD and abTCR-7 in T cells using ananti-FLAG antibody to detect the FLAG-tagged constructs. Staining forCD3 was included as a loading control. FIG. 16C shows killing ofSK-HEP-1 and SK-HEP-1-AFP-MG cells mediated by T cells transduced witheither the abTCR-6MD or abTCR-7.

FIG. 17 shows killing of cancer cell lines JeKo-1, IM9, THP-1 andJurkat, mediated by mock-transduced T cells or T cells transduced witheither a CAR or an abTCR-6MD, both having the same anti-CD19 bindingmoiety variable domains.

FIGS. 18A and 18B show the level of secretion of a panel of cytokines bymock-transduced T cells or T cells transduced with either a CAR or anabTCR-6MD, both having the same anti-CD19 binding moiety variabledomains, co-cultured with JeKo-1, IM9, THP-1 or Jurkat cell lines.

FIGS. 19A-19C show target-specific activation of cytokine expression inCD4⁺ T cells transduced with an anti-CD19 abTCR and incubated withcancer cell lines positive or negative for CD19 expression.

FIG. 20 shows flow cytometry analysis of T cell differentiation markersCD28, CCR7 and granzyme B on CAR- or abTCR-transduced T cells, bothhaving the same anti-CD19 binding moiety variable domains, upon exposureto antigen-positive or -negative target cells.

FIG. 21 shows proliferation of CAR- or abTCR-transduced CD4⁺ or CD8⁺ Tcells, both chimeric receptors having the same anti-CD19 binding moietyvariable domains, during exposure to antigen-positive target cells, asassessed by dye dilution from day 2 to day 3 following initiation ofexposure.

FIG. 22 shows internalization of chimeric receptors on CAR- orabTCR-transduced T cells, both chimeric receptors having the sameanti-CD19 binding moiety variable domains, at the indicated time pointsas assessed by flow cytometry analysis of cells stained for surfacechimeric receptors with an anti-idiotype antibody targeting theanti-CD19 binding moiety.

FIGS. 23A and 23B show the characterization of T cells transduced witheither an abTCR (anti-CD19 abTCR-6MD) or cTCR (anti-CD19-cTCR), bothhaving the same anti-CD19 binding moiety variable domains. FIG. 23Ashows cell growth of the abTCR and cTCR T cells. FIG. 23B shows killingof CD19-positive cancer cell line Nalm-6 mediated by mock-transduced Tcells or T cells transduced with either the abTCR or cTCR.

FIG. 24 shows killing of cancer cell lines IM9, Colo205, MDA-231, MCF7,JeKo-1, Raji, Hep1, and Jurkat, mediated by mock-transduced T cells or Tcells transduced with either a CAR (#35 CAR) or an abTCR-6MD (#35abTCR), both having the same anti-NY-ESO-1 binding moiety variabledomains.

FIG. 25A shows flow cytometry analysis of the expression of CD3 and CD56on a subset of NKT cells purified from human PBMCs.

FIG. 25B shows the level of secretion of cytokines IL-2, GM-CSF, IFNγ,and TNFα by mock-transduced T cells or T cells transduced with anabTCR-6MD having an anti-CD19 binding moiety, co-cultured with Raji orRaji-CD19ko cell lines. Controls included mock-transduced orabTCR-transduced T cells alone, and Raji or Raji-CD19ko cells alone.

FIG. 26A shows flow cytometry analysis of the expression of CD25 and CD4on a subset of Treg cells purified from human PBMCs.

FIG. 26B shows the level of secretion of cytokines IL-2, GM-CSF, IFNγ,and TNFα by mock-transduced T cells or T cells transduced with anabTCR-6MD having an anti-CD19 binding moiety, co-cultured with Raji orRaji-CD19ko cell lines.

FIG. 27 shows killing of cancer cell lines HepG2, SK-Hep1, andSK-Hep1-AFP MG, mediated by mock-transduced T cells or T cellstransduced with abTCRs having various immunoglobulin CH1 domains, eachhaving the same anti-AFP binding moiety.

FIG. 28 shows a schematic representation of the various abTCR constructdesigns containing one or more co-stimulatory domains (abTCR-6M-1,abTCR-6M-2, abTCR-6M-3, abTCR-6M-4, abTCR-6M-5, abTCR-6M-6, abTCR-6M-7,abTCR-6M-8).

FIG. 29 shows killing of cancer cell lines HepG2, SK-Hep1, andSK-Hep1-AFP MG, mediated by mock-transduced T cells or T cellstransduced with various abTCRs having one or more C-terminalco-stimulatory domains, each having the same anti-AFP binding moiety.

FIG. 30 shows the level of secretion of cytokines IL-2, GM-CSF, IFNγ,and TNFα by mock-transduced T cells or T cells transduced with variousabTCRs having one or more C-terminal co-stimulatory domains, each havingthe same anti-AFP binding moiety, co-cultured with SK-Hep1 orSK-Hep1-AFP MG cell lines.

FIG. 31 shows killing of cancer cell lines Raji, Raji-CD19ko, andJeKo-1, mediated by mock-transduced T cells or T cells transduced withvarious abTCRs having one or more C-terminal co-stimulatory domains,each having the same anti-CD19 binding moiety.

FIG. 32 shows the level of secretion of cytokines IL-2, GM-CSF, IFNγ,and TNFα by mock-transduced T cells or T cells transduced with variousabTCRs having one or more C-terminal co-stimulatory domains, each havingthe same anti-CD19 binding moiety, co-cultured with Raji, Raji-CD19ko,or JeKo-1 cells.

FIG. 33 shows the body weight change over time in a subcutaneous mousexenograft model of SK-HEP-1-AFP-MG treated with intravenous injection ofeither mock-transduced T cells or T cells transduced with an abTCR-6MDhaving an anti-AFP158/HLA-A*02:01 binding moiety.

FIG. 34A shows the tumor growth in a subcutaneous mouse model ofSK-HEP-1-AFP-MG treated with intravenous injection of eithermock-transduced T cells or T cells transduced with an abTCR-6MD havingan anti-AFP158/HLA-A*02:01 binding moiety.

FIG. 34B shows the tumor growth in a subcutaneous mouse model ofSK-HEP-1-AFP-MG with no treatment or with a single intratumoralinjection of T cells transduced with an abTCR-6MD having ananti-AFP158/HLA-A*02:01 binding moiety when the average tumor volumereached 300 mm³.

FIG. 35 shows tumor growth in reporter Raji intravenous xenograft micetreated with T cells transduced with various anti-CD19 abTCRs (clones 5,5-3, 5-9, and 5-14). Mock-transduced T-cells and no T cell treatmentwere included as controls.

FIG. 36 shows the serum level of IL-2, IFN-γ, TNF-α, and IL-10 in Rajixenograft mice injected with mock-transduced T cells or T cellstransduced with either a CAR or an abTCR-6MD, both having the Clone 5-13anti-CD19 binding moiety variable domains.

FIG. 37 shows quantitation of tumor growth in reporter Raji xenograftmice treated with T cells transduced with either a CAR or an abTCR-6MD,both having the Clone 5-13 anti-CD19 binding moiety variable domains.Mock-transduced T-cells were included as controls.

FIG. 38 shows imaging results for tumor-derived bioluminescence inreporter Raji xenograft mice treated with T cells transduced with eithera CAR or an abTCR-6MD, both having the Clone 5-13 anti-CD19 bindingmoiety variable domains. Mock-transduced T-cells were included ascontrols. The grey-scale converted heatmap indicates total photons persecond at the location of tumors, which appear as dark spots overlaid onthe mouse images.

FIG. 39 shows quantitation of tumor growth in reporter Raji xenograftmice re-challenged with tumor cells 7 weeks following initial tumor cellimplantation and treatment with T cells transduced with Clone 5-13anti-CD19 abTCR-6MD. Mock-transduced T-cells were included as controls.

FIG. 40 shows tumor growth in reporter NALM-6 intravenous xenograft micetreated with T cells transduced with either a CAR or an abTCR-6MD, bothhaving the Clone 5-13 anti-CD19 binding moiety variable domains.Mock-transduced T-cells and no T cell treatment were included ascontrols.

FIG. 41 shows the serum level of IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ,and TNF-α in NALM-6 xenograft mice injected with cells transduced witheither a CAR or an abTCR-6MD, both having the Clone 5-13 anti-CD19binding moiety variable domains. Mock-transduced T-cells and no T celltreatment were included as controls.

FIG. 42 shows the amount of chimeric receptor-positive T cells in bloodfrom NALM-6 xenograft mice injected with cells transduced with either aCAR or an abTCR-6MD, both having the Clone 5-13 anti-CD19 binding moietyvariable domains, at 7 and 13 days post-treatment.

FIG. 43 shows flow cytometry analysis for tumor cells in blood fromNALM-6 xenograft mice injected with cells transduced with either a CARor an abTCR-6MD, both having the Clone 5-13 anti-CD19 binding moietyvariable domains, at 13 days post-treatment.

FIG. 44 shows flow cytometry analysis for tumor cells in bone marrowfrom NALM-6 xenograft mice injected with cells transduced with either aCAR or an abTCR-6MD, both having the Clone 5-13 anti-CD19 binding moietyvariable domains, at 13 days post-treatment.

FIG. 45 shows flow cytometry analysis for PD-1 expression on CD3⁺ Tcells that are either CD4⁺ or CD8⁺ in blood from NALM-6 xenograft miceinjected with cells transduced with either a CAR or an abTCR-6MD, bothhaving the Clone 5-13 anti-CD19 binding moiety variable domains.

FIG. 46 shows flow cytometry analysis for PD-1 expression on CD3⁺ Tcells that are either CD4⁺ or CD8⁺ in bone marrow from NALM-6 xenograftmice injected with cells transduced with either a CAR or an abTCR-6MD,both having the Clone 5-13 anti-CD19 binding moiety variable domains.

DETAILED DESCRIPTION OF THE INVENTION

The present application provides an isolated chimeric antibody/T cellreceptor construct (referred to herein as “abTCR”) that comprises a) anantibody moiety, such as a Fab or FAT fragment, that specifically bindsto a target antigen; and b) a T cell receptor module (TCRM) capable ofrecruiting at least one TCR-associated signaling module.

We have developed a series of novel and synthetic chimeric antibody/TCRconstructs that combine the binding specificity and affinity of ourTCR-like mAbs, as well as conventional mAbs, with the target-specificcytotoxic potency and controlled activation afforded by TCRs. Primary Tcells transduced to express abTCRs showed efficient surface expressionand formation of stable TCR-like signaling complexes in association withendogenous CD3 molecules. When engineered into T cells, the abTCRsendowed the T cells with potent cytotoxicity against target-bearingtumor cells both in vitro and in vivo, in both MHC-dependent(peptide/MHC antigen) and MHC-independent (cell-surface antigen)configurations. Target-specific activation was observed for multipledifferent T cell subsets transduced to express an abTCR, including CD4+T cells, CD8+ T cells, natural killer T (NKT) cells, and regulatory T(Treg) cells. In addition, abTCRs including intracellular co-stimulatorysequences were found to perform as well as, and in some cases betterthan, corresponding abTCRs without any co-stimulatory sequences.

Despite the remarkable curative potential demonstrated with CAR T celltherapy, clinical trials continue to trigger severe adverse events thatare associated with excessive cytokine release and uncontrolled T-cellproliferation. Without being bound by theory, it is believed that abTCRscan be regulated by the naturally occurring machinery that controls TCRactivation, requiring assembly with an endogenous CD3 complex toactivate T-cell-mediated killing, and can thus avoid beingconstitutively activated. We have found that T cells transduced withabTCR constructs express lower levels of cytokines (e.g., IL-2) and Tcell exhaustion markers (e.g., PD-1, TIM3, and LAG1) than T cellstransduced with corresponding chimeric antigen receptors (CARs) bearingthe same antibody variable regions, while having equivalent potency incancer cell killing. This strategy thus provides a significant technicaladvantage over using CARs, yielding T cells whose cytotoxic signalingresponds to endogenous T-cell regulatory mechanisms and which have thepotential to functionally persist longer in vivo. By combining theexquisitely optimized binding of monoclonal antibodies to specificantigens, such as cell surface antigens or peptide/MHC complexes, withthe ability of the T cell receptor to engage endogenous signalingcomplexes to activate immune cells, the invention allows for highlyspecific and potent targeting of low-copy number cell surface antigens,as well as intracellular or secreted antigens via peptide/MHC complexes.

The present application thus provides an abTCR (such as an isolatedabTCR) comprising an antibody moiety that specifically binds to a targetantigen and a TCRM capable of recruiting at least one TCR-associatedsignaling module. The abTCR may be a heterodimer comprising a firstpolypeptide chain and a second polypeptide chain. The antibody moietymay comprise a heavy chain variable antibody domain (V_(H)) and a lightchain variable antibody domain (V_(L)). In some embodiments, theantibody moiety further comprises one or more antibody heavy chainconstant domains, such as a heavy chain constant 1 antibody domain(C_(H)1) and/or a light chain constant antibody domain (C_(L)). The TCRMcomprises a first T cell receptor domain (TCRD) comprising atransmembrane domain of a first TCR subunit and a second TCRD comprisinga transmembrane domain of a second TCR subunit. The first polypeptidechain and the second polypeptide chain of the abTCR may be linked viaone or more disulfide bonds. See FIG. 1A for exemplary abTCR constructdesigns.

In another aspect, there is provided one or more nucleic acids encodingan abTCR.

In yet another aspect, there is provided a complex (referred to hereinas an “abTCR-CD3 complex”) comprising an abTCR and at least oneTCR-associated signaling module. The complex may be an octamercomprising the four dimers abTCR, CD3δε, CD3γε, and ζζ. Also provided isan effector cell, such as a T cell, expressing or associated with anabTCR or abTCR-CD3 complex.

In yet another aspect, there is provided a composition comprising anabTCR. The composition can be a pharmaceutical composition comprising anabTCR or an effector cell expressing or associated with the abTCR (forexample a T cell expressing an abTCR).

Also provided are methods of making and using an abTCR (or cellsexpressing or associated with an abTCR) for treatment purposes, as wellas kits and articles of manufacture useful for such methods. Furtherprovided are methods of treating a disease using an abTCR (or cellsexpressing or associated with an abTCR).

Definitions

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results, including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: alleviating one ormore symptoms resulting from the disease, diminishing the extent of thedisease, stabilizing the disease (e.g., preventing or delaying theworsening of the disease), preventing or delaying the spread (e.g.,metastasis) of the disease, preventing or delaying the recurrence of thedisease, delay or slowing the progression of the disease, amelioratingthe disease state, providing a remission (partial or total) of thedisease, decreasing the dose of one or more other medications requiredto treat the disease, delaying the progression of the disease,increasing or improving the quality of life, increasing weight gain,and/or prolonging survival. Also encompassed by “treatment” is areduction of pathological consequence of the disease (such as, forexample, tumor volume in cancer). The methods of the inventioncontemplate any one or more of these aspects of treatment.

The terms “recurrence,” “relapse” or “relapsed” refers to the return ofa cancer or disease after clinical assessment of the disappearance ofdisease. A diagnosis of distant metastasis or local recurrence can beconsidered a relapse.

The term “refractory” or “resistant” refers to a cancer or disease thathas not responded to treatment.

“Activation”, as used herein in relation to T cells, refers to the stateof a T cell that has been sufficiently stimulated to induce detectablecellular proliferation. Activation can also be associated with inducedcytokine production, and detectable effector functions.

The term “antibody” or “antibody moiety” includes full-length antibodiesand antigen-binding fragments thereof. A full-length antibody comprisestwo heavy chains and two light chains. The variable regions of the lightand heavy chains are responsible for antigen-binding. The variablesregion in both chains generally contain three highly variable loopscalled the complementarity determining regions (CDRs) (light chain (LC)CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRsincluding HC-CDR1, HC-CDR2, and HC-CDR3). CDR boundaries for theantibodies and antigen-binding fragments disclosed herein may be definedor identified by the conventions of Kabat, Chothia, or Al-Lazikani(Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987;Kabat 1991). The three CDRs of the heavy or light chains are interposedbetween flanking stretches known as framework regions (FRs), which aremore highly conserved than the CDRs and form a scaffold to support thehypervariable loops. The constant regions of the heavy and light chainsare not involved in antigen-binding, but exhibit various effectorfunctions. Antibodies are assigned to classes based on the amino acidsequence of the constant region of their heavy chain. The five majorclasses or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, whichare characterized by the presence of α, δ, ε, γ, and μ heavy chains,respectively. Several of the major antibody classes are divided intosubclasses such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3(γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2(α2 heavy chain).

The term “antigen-binding fragment” as used herein refers to an antibodyfragment including, for example, a diabody, a Fab, a Fab′, a F(ab′)2, anFv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, abispecific dsFv (dsFv-dsFv′), a disulfide stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), an scFv dimer(bivalent diabody), a multispecific antibody formed from a portion of anantibody comprising one or more CDRs, a camelized single domainantibody, a nanobody, a domain antibody, a bivalent domain antibody, orany other antibody fragment that binds to an antigen but does notcomprise a complete antibody structure. An antigen-binding fragment iscapable of binding to the same antigen to which the parent antibody or aparent antibody fragment (e.g., a parent scFv) binds. In someembodiments, an antigen-binding fragment may comprise one or more CDRsfrom a particular human antibody grafted to a framework region from oneor more different human antibodies.

A “Fab-like antigen-binding module” refers to an antibody moiety thatcomprises a first polypeptide chain and a second polypeptide chain,wherein the first and second polypeptide chains comprise a V_(L)antibody domain, a C_(L) antibody domain, a V_(H) antibody domain, and aC_(H)1 antibody domain. The V_(L) and C_(L) antibody domains may be onone chain with the V_(H) and C_(H)1 antibody domains on the other chain,or the V_(L) and C_(H)1 antibody domains may be on one chain with theV_(H) and C_(L) antibody domains on the other chain. In someembodiments, the first and second polypeptide chains are linked by adisulfide bond.

As used herein, a first antibody moiety “competes” for binding to atarget antigen with a second antibody moiety when the first antibodymoiety inhibits target antigen-binding of the second antibody moiety byat least about 50% (such as at least about any of 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98% or 99%) in the presence of an equimolarconcentration of the first antibody moiety, or vice versa. A highthroughput process for “binning” antibodies based upon theircross-competition is described in PCT Publication No. WO 03/48731.

As use herein, the term “specifically binds” or “is specific for” refersto measurable and reproducible interactions, such as binding between atarget and an antibody or antibody moiety, that is determinative of thepresence of the target in the presence of a heterogeneous population ofmolecules, including biological molecules. For example, an antibodymoiety that specifically binds to a target (which can be an epitope) isan antibody moiety that binds the target with greater affinity, avidity,more readily, and/or with greater duration than its bindings to othertargets. In some embodiments, an antibody moiety that specifically bindsto an antigen reacts with one or more antigenic determinants of theantigen (for example a cell surface antigen or a peptide/MHC proteincomplex) with a binding affinity that is at least about 10 times itsbinding affinity for other targets.

The term “T cell receptor,” or “TCR,” refers to a heterodimeric receptorcomposed of αβ or γδ chains that pair on the surface of a T cell. Eachα, β, γ, and δ chain is composed of two Ig-like domains: a variabledomain (V) that confers antigen recognition through the complementaritydetermining regions (CDR), followed by a constant domain (C) that isanchored to cell membrane by a connecting peptide and a transmembrane(TM) region. The TM region associates with the invariant subunits of theCD3 signaling apparatus. Each of the V domains has three CDRs. TheseCDRs interact with a complex between an antigenic peptide bound to aprotein encoded by the major histocompatibility complex (pMHC) (Davisand Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu RevImmunol, 16, 523-544; Murphy (2012), xix, 868 p).

The term “TCR-associated signaling module” refers to a molecule having acytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) thatis part of the TCR-CD3 complex. TCR-associated signaling modules includeCD3γε, CD3δε, and ζζ.

The term “module” when referring to a protein or portion of a proteinmeans the protein or portion of the protein comprises a plurality ofpolypeptide chains (e.g., a dimeric protein or portion of a dimericprotein). The plurality of polypeptide chains may be linked, such as bya linker (e.g., a peptide linker) or chemical linkage (e.g., a peptidelinkage). A “module” is meant to include structurally and/orfunctionally related portions of one or more polypeptides which make upthe protein. For example, a transmembrane module of a dimeric receptormay refer to the portions of each polypeptide chain of the receptor thatspan the membrane. A module may also refer to related portions of asingle polypeptide chain. For example, a transmembrane module of amonomeric receptor may refer to portions of the single polypeptide chainof the receptor that span the membrane.

The term “T cell receptor module,” or “TCRM,” refers to a heterodimercomprising sequences derived from a T cell receptor. The TCRM comprisesT cell receptor transmembrane domains, and may further comprise all or aportion of T cell receptor connecting peptides and/or intracellulardomains.

An “isolated” construct (such as an abTCR) as used herein refers to aconstruct that (1) is not associated with proteins found in nature, (2)is free of other proteins from the same source, (3) is expressed by acell from a different species, or, (4) does not occur in nature.

The term “isolated nucleic acid” as used herein is intended to mean anucleic acid of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated nucleic acid” (1)is not associated with all or a portion of a polynucleotide in which the“isolated nucleic acid” is found in nature, (2) is operably linked to apolynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

As used herein, the term “CDR” or “complementarity determining region”is intended to mean the non-contiguous antigen combining sites foundwithin the variable region of both heavy and light chain polypeptides.These particular regions have been described by Kabat et al., J. Biol.Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and HumanServices, “Sequences of proteins of immunological interest” (1991); byChothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al.,J. Mol. Biol. 262:732-745 (1996), where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or grafted antibodies or variants thereof is intended tobe within the scope of the term as defined and used herein. The aminoacid residues which encompass the CDRs as defined by each of the abovecited references are set forth below in Table 1 as a comparison.

TABLE 1 CDR DEFINITIONS Kabat¹ Chothia² MacCallum³ V_(H) CDR1 31-3526-32 30-35 V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3  95-102  96-101 93-101 V_(L) CDR1 24-34 26-32 30-36 V_(L) CDR2 50-56 50-52 46-55 V_(L)CDR3 89-97 91-96 89-96 ¹Residue numbering follows the nomenclature ofKabat et al., supra ²Residue numbering follows the nomenclature ofChothia et al., supra ³Residue numbering follows the nomenclature ofMacCallum et al., supra

The term “chimeric antibodies” refer to antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit a biological activity of thisinvention (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.Acad. Sci. USA, 81:6851-6855 (1984)).

The term “semi-synthetic” in reference to an antibody or antibody moietymeans that the antibody or antibody moiety has one or more naturallyoccurring sequences and one or more non-naturally occurring (i.e.,synthetic) sequences.

The term “fully synthetic” in reference to an antibody or antibodymoiety means that the antibody or antibody moiety has fixed, mostly orall naturally occurring V_(H)/V_(L) framework pairings, butnon-naturally occurring (i.e., synthetic) sequences of all 6 CDRs ofboth heavy and light chains. Non-naturally occurring CDRs include thosecomprising modified human CDR sequences, such as CDR sequences modifiedby conservative amino acid substitutions or introduced cysteineresidues.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region (HVR) of the recipient are replaced by residuesfrom a hypervariable region of a non-human species (donor antibody) suchas mouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies cancomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

“Homology” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are“homologous” at that position. The “percent of homology” or “percentsequence identity” between two sequences is a function of the number ofmatching or homologous positions shared by the two sequences divided bythe number of positions compared times 100, considering any conservativesubstitutions as part of the sequence identity. For example, if 6 of 10of the positions in two sequences are matched or homologous then the twosequences are 60% homologous. By way of example, the DNA sequencesATTGCC and TATGGC share 50% homology. Generally, a comparison is madewhen two sequences are aligned to give maximum homology. Alignment forpurposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are generated using thesequence comparison computer program MUSCLE (Edgar, R. C., Nucleic AcidsResearch 32(5):1792-1797, 2004; Edgar, R. C., BMC Bioinformatics5(1):113, 2004).

The “C_(H)1 domain” of a human IgG (also referred to as “C1” of “H1”domain) usually extends from about amino acid 118 to about amino acid215 (EU numbering system).

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “inducible promoter” refers to a promoter whose activity can beregulated by adding or removing one or more specific signals. Forexample, an inducible promoter may activate transcription of an operablylinked nucleic acid under a specific set of conditions, e.g., in thepresence of an inducing agent that activates the promoter and/orrelieves repression of the promoter.

An “effective amount” of an abTCR or composition comprising an abTCR asdisclosed herein is an amount sufficient to carry out a specificallystated purpose. An “effective amount” can be determined empirically andby known methods relating to the stated purpose.

The term “therapeutically effective amount” refers to an amount of anabTCR or composition comprising an abTCR as disclosed herein, effectiveto “treat” a disease or disorder in an individual. In the case ofcancer, the therapeutically effective amount of an abTCR or compositioncomprising an abTCR as disclosed herein can reduce the number of cancercells; reduce the tumor size or weight; inhibit (i.e., slow to someextent and preferably stop) cancer cell infiltration into peripheralorgans; inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Tothe extent an abTCR or composition comprising an abTCR as disclosedherein can prevent growth and/or kill existing cancer cells, it can becytostatic and/or cytotoxic. In some embodiments, the therapeuticallyeffective amount is a growth inhibitory amount. In some embodiments, thetherapeutically effective amount is an amount that improves progressionfree survival of a patient. In the case of infectious disease, such asviral infection, the therapeutically effective amount of an abTCR orcomposition comprising an abTCR as disclosed herein can reduce thenumber of cells infected by the pathogen; reduce the production orrelease of pathogen-derived antigens; inhibit (i.e., slow to some extentand preferably stop) spread of the pathogen to uninfected cells; and/orrelieve to some extent one or more symptoms associated with theinfection. In some embodiments, the therapeutically effective amount isan amount that extends the survival of a patient.

As used herein, by “pharmaceutically acceptable” or “pharmacologicallycompatible” is meant a material that is not biologically or otherwiseundesirable, e.g., the material may be incorporated into apharmaceutical composition administered to a patient without causing anysignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. Pharmaceutically acceptable carriers orexcipients have preferably met the required standards of toxicologicaland manufacturing testing and/or are included on the Inactive IngredientGuide prepared by the U.S. Food and Drug administration.

It is understood that embodiments of the invention described hereininclude “consisting” and/or “consisting essentially of” embodiments.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein, reference to “not” a value or parameter generally meansand describes “other than” a value or parameter. For example, the methodis not used to treat cancer of type X means the method is used to treatcancer of types other than X.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

Chimeric Antibody/T Cell Receptor Constructs

In one aspect, the present invention provides a target antigen-specificchimeric antibody/T cell receptor (abTCR) that specifically binds to atarget antigen (such as a cell surface antigen or a peptide/MHC complex)and is capable of recruiting at least one TCR-associated signalingmodule (such as CD3δε, CD3γε, or ζζ). In some embodiments, the abTCRcomprises a first polypeptide chain and a second polypeptide chain. Insome embodiments, the first and second polypeptide chains are linked,such as by a covalent linkage (e.g., peptide or other chemical linkage)or non-covalent linkage. In some embodiments, the abTCR is a heterodimercomprising a first polypeptide chain and a second polypeptide chain. Insome embodiments, the first polypeptide chain and the second polypeptidechain are linked by at least one disulfide bond. The specificity of theabTCR derives from an antibody moiety that confers binding specificityto the target antigen. In some embodiments, the antibody moiety is aFab-like antigen-binding module comprising V_(H), C_(H)1, V_(L), andC_(L) antibody domains. In some embodiments, the antibody moiety is anFv-like antigen-binding module comprising V_(H) and V_(L) antibodydomains. The capability of the abTCR to recruit a TCR-associatedsignaling module derives from a T cell receptor module (TCRM). In someembodiments, the TCRM comprises the transmembrane module of a TCR (suchas an αβTCR or a γδTCR). In some embodiments, the TCRM further comprisesone or both of the connecting peptides or fragments thereof of a TCR. Insome embodiments, the transmembrane module and the connecting peptidesor fragments thereof are derived from the same TCR type (αβ or γδ). Insome embodiments, the transmembrane module is derived from an αβ TCR andthe connecting peptides or fragments thereof are derived from a γδ TCR,or the transmembrane module is derived from a γδ TCR and the connectingpeptides or fragments thereof are derived from an αβ TCR. In someembodiments, the abTCR further comprises at least one intracellulardomain. In some embodiments, one or more of the at least oneintracellular domain of the abTCR comprises a sequence from theintracellular domain of a TCR. In some embodiments, one or more of theat least one intracellular domain of the abTCR comprises a T cellcostimulatory signaling sequence. The costimulatory signaling sequencecan be a portion of the intracellular domain of a costimulatory moleculeincluding, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like.In some embodiments, the antibody moiety is contained in anextracellular domain of the abTCR. In some embodiments, the abTCRfurther comprises one or more peptide linkers between the antibodymoiety and the TCRM to optimize the length of the extracellular domain.In some embodiments, reference to an antigen-binding module (such as aFab-like or Fv-like antigen-binding module) that specifically binds to atarget antigen means that the antigen-binding module binds to the targetantigen with a) an affinity that is at least about 10 (including forexample at least about any of 10, 20, 30, 40, 50, 75, 100, 200, 300,400, 500, 750, 1000 or more) times its binding affinity for othermolecules; or b) a K_(d) no more than about 1/10 (such as no more thanabout any of 1/10, 1/20, 1/30, 1/40, 1/50, 1/75, 1/100, 1/200, 1/300,1/400, 1/500, 1/750, 1/1000 or less) times its K_(d) for binding toother molecules. Binding affinity can be determined by methods known inthe art, such as ELISA, fluorescence activated cell sorting (FACS)analysis, or radioimmunoprecipitation assay (RIA). K_(d) can bedetermined by methods known in the art, such as surface plasmonresonance (SPR) assay utilizing, for example, Biacore instruments, orkinetic exclusion assay (KinExA) utilizing, for example, Sapidyneinstruments.

Contemplated abTCR constructs include, for example, abTCRs thatspecifically bind to cell surface antigens and abTCRs that specificallybind to cell surface-presented peptide/MHC complexes.

In some embodiments, the abTCR comprises a Fab-like antigen-bindingmodule comprising a) a first polypeptide chain comprising a firstantigen-binding domain comprising a V_(H) antibody domain and a C_(H)1antibody domain and b) a second polypeptide chain comprising a secondantigen-binding domain comprising a V_(L) antibody domain and a C_(L)antibody domain. In some embodiments, the first antigen-binding domaincomprises the V_(H) antibody domain amino-terminal to the C_(H)1antibody domain and/or the second antigen-binding domain comprises theV_(L) antibody domain amino-terminal to the C_(L) antibody domain. Insome embodiments, there is a peptide linker between the V_(L) and C_(L)antibody domains and/or a peptide linker between the V_(H) and C_(H)1antibody domains. In some embodiments, all of the V_(L) antibody domainand V_(H) antibody domain CDRs are derived from the same antibodymoiety. In some embodiments, the V_(L) antibody domain and the V_(H)antibody domain comprise antibody CDRs derived from more than oneantibody moiety. In some embodiments, the V_(L) antibody domaincomprises antibody CDRs derived from a V_(H) antibody domain and/or theV_(H) antibody domain comprises antibody CDRs derived from a V_(L)antibody domain. In some embodiments, the V_(L) antibody domaincomprises framework regions derived from one antibody and one or moreCDRs derived from another antibody and/or the V_(H) antibody domaincomprises framework regions derived from one antibody and one or moreCDRs derived from another antibody. In some embodiments, the first andsecond polypeptide chains are linked, such as by a covalent linkage(e.g., peptide or other chemical linkage) or non-covalent linkage. Insome embodiments, the first and second antigen-binding domains arelinked by a disulfide bond. In some embodiments, the first and secondantigen-binding domains are linked by a disulfide bond between a residuein the C_(H)1 domain and a residue in the C_(L) domain. In someembodiments, the C_(H)1 domain is derived from an IgG (e.g, IgG1, IgG2,IgG3, or IgG4), IgA (e.g., IgA1 or IgA2), IgD, IgM, or IgE heavy chain,optionally human. In some embodiments, the C_(H)1 domain comprises (suchas consists of) the amino acid sequence of any one of SEQ ID NOs: 39 and60-69). In some embodiments, the C_(H)1 domain is a variant comprisingone or more modifications (e.g., amino acid substitutions, insertions,and/or deletions) compared to the sequence from which it is derived. Insome embodiments, the C_(L) domain is derived from a kappa or lambdalight chain, optionally human. In some embodiments, the C_(L) domaincomprises (such as consists of) the amino acid sequence of SEQ ID NO:41. In some embodiments, the C_(L) domain is a variant comprising one ormore modifications (e.g., amino acid substitutions, insertions, and/ordeletions) compared to the sequence from which it is derived. In someembodiments, the C_(H)1 and/or C_(L) domains comprise one or moremodifications that do not substantially alter their binding affinitiesfor one another. In some embodiments, the C_(H)1 and/or C_(L) domainscomprise one or more modifications that increase their bindingaffinities for one another and/or introduce a non-naturally occurringdisulfide bond. In some embodiments, the C_(H)1 and C_(L) domainscomprise a knob-into-hole modification (see, for example, Carter P. JImmunol Methods. 248:7-15, 2001). In some embodiments, the C_(H)1 andC_(L) domains are modified by electrostatic steering to enhance theirassociation with one another (see, for example, WO2006106905 andGunasekaran K, et al. J Biol Chem. 285:19637-46, 2010). In someembodiments, the Fab-like antigen-binding module is human, humanized,chimeric, semi-synthetic, or fully synthetic.

In some embodiments, the abTCR comprises a Fab-like antigen-bindingmodule comprising a) a first polypeptide chain comprising a firstantigen-binding domain comprising a V_(L) antibody domain and a C_(H)1antibody domain and b) a second polypeptide chain comprising a secondantigen-binding domain comprising a V_(H) antibody domain and a C_(L)antibody domain. In some embodiments, the first antigen-binding domaincomprises the V_(L) antibody domain amino-terminal to the C_(H)1antibody domain and/or the second antigen-binding domain comprises theV_(H) antibody domain amino-terminal to the C_(L) antibody domain. Insome embodiments, there is a peptide linker between the V_(H) and C_(L)antibody domains and/or a peptide linker between the V_(L) and C_(H)1antibody domains. In some embodiments, all of the V_(L) antibody domainand V_(H) antibody domain CDRs are derived from the same antibodymoiety. In some embodiments, the V_(L) antibody domain and the V_(H)antibody domain comprise antibody CDRs derived from more than oneantibody moiety. In some embodiments, the V_(L) antibody domaincomprises antibody CDRs derived from a V_(H) antibody domain and/or theV_(H) antibody domain comprises antibody CDRs derived from a V_(L)antibody domain. In some embodiments, the V_(L) antibody domaincomprises framework regions derived from one antibody and one or moreCDRs derived from another antibody and/or the V_(H) antibody domaincomprises framework regions derived from one antibody and one or moreCDRs derived from another antibody. In some embodiments, the first andsecond polypeptide chains are linked, such as by a covalent linkage(e.g., peptide or other chemical linkage) or non-covalent linkage. Insome embodiments, the first and second antigen-binding domains arelinked by a disulfide bond. In some embodiments, the first and secondantigen-binding domains are linked by a disulfide bond between a residuein the C_(H)1 domain and a residue in the C_(L) domain. In someembodiments, the C_(H)1 domain is derived from an IgG (e.g, IgG1, IgG2,IgG3, or IgG4), IgA (e.g., IgA1 or IgA2), IgD, IgM, or IgE heavy chain,optionally human. In some embodiments, the C_(H)1 domain comprises (suchas consists of) the amino acid sequence of any one of SEQ ID NOs: 39 and60-69). In some embodiments, the C_(H)1 domain is a variant comprisingone or more modifications (e.g., amino acid substitutions, insertions,and/or deletions) compared to the sequence from which it is derived. Insome embodiments, the C_(L) domain is derived from a kappa or lambdalight chain, optionally human. In some embodiments, the C_(L) domaincomprises (such as consists of) the amino acid sequence of SEQ ID NO:41. In some embodiments, the C_(L) domain is a variant comprising one ormore modifications (e.g., amino acid substitutions, insertions, and/ordeletions) compared to the sequence from which it is derived. In someembodiments, the C_(H)1 and/or C_(L) domains comprise one or moremodifications that do not substantially alter their binding affinitiesfor one another. In some embodiments, the C_(H)1 and/or C_(L) domainscomprise one or more modifications that increase their bindingaffinities for one another and/or introduce a non-naturally occurringdisulfide bond. In some embodiments, the C_(H)1 and C_(L) domainscomprise a knob-into-hole modification (see, for example, Carter P. JImmunol Methods. 248:7-15, 2001). In some embodiments, the C_(H)1 andC_(L) domains are modified by electrostatic steering to enhance theirassociation with one another (see, for example, WO2006106905 andGunasekaran K, et al. J Biol Chem. 285:19637-46, 2010). In someembodiments, the Fab-like antigen-binding module is human, humanized,chimeric, semi-synthetic, or fully synthetic.

In some embodiments, the abTCR comprises an Fv-like antigen-bindingmodule comprising a) a first polypeptide chain comprising a firstantigen-binding domain comprising a V_(H) antibody domain and b) asecond polypeptide chain comprising a second antigen-binding domaincomprising a V_(L) antibody domain. In some embodiments, there is afirst peptide linker fused to the C-terminus of the V_(L) antibodydomain and/or a second peptide linker fused to the C-terminus of theV_(H) antibody domain. In some embodiments, the first and second peptidelinkers are capable of binding to one another. In some embodiments, thefirst and/or second peptide linkers are derived from immunoglobulinheavy and/or light chain constant regions. In some embodiments, thefirst and/or second peptide linkers comprise a CH3 antibody domain or avariant thereof. In some embodiments, immunoglobulin heavy chainconstant domains (e.g., C_(H)1 or CH3) contained in the peptide linkersare derived from an IgG (e.g, IgG1, IgG2, IgG3, or IgG4), IgA (e.g.,IgA1 or IgA2), IgD, IgM, or IgE heavy chain, optionally human. In someembodiments, the first and/or second peptide linkers are derived fromTCR subunit constant regions. For example, in some embodiments, thefirst and/or second peptide linkers are derived from a) TCR α and βsubunit constant domains; or b) TCR γ and δ subunit constant domains. Insome embodiments, the first and/or second peptide linkers are synthetic.In some embodiments, all of the V_(L) antibody domain and V_(H) antibodydomain CDRs are derived from the same antibody moiety. In someembodiments, the V_(L) antibody domain and the V_(H) antibody domaincomprise antibody CDRs derived from more than one antibody moiety. Insome embodiments, the V_(L) antibody domain comprises antibody CDRsderived from a V_(H) antibody domain and/or the V_(H) antibody domaincomprises antibody CDRs derived from a V_(L) antibody domain. In someembodiments, the V_(L) antibody domain comprises framework regionsderived from one antibody and one or more CDRs derived from anotherantibody and/or the V_(H) antibody domain comprises framework regionsderived from one antibody and one or more CDRs derived from anotherantibody. In some embodiments, the first and second polypeptide chainsare linked, such as by a covalent linkage (e.g., peptide or otherchemical linkage) or non-covalent linkage. In some embodiments, thefirst and second antigen-binding domains are linked by a disulfide bond.In some embodiments, the first and second peptide linkers are linked bya disulfide bond. In some embodiments, the first and/or second peptidelinker is a variant comprising one or more modifications (e.g., aminoacid substitutions, insertions, and/or deletions) compared to thesequence from which it is derived. In some embodiments, the first and/orsecond peptide linkers comprise one or more modifications that do notsubstantially alter their binding affinity for one another. In someembodiments, the first and/or second peptide linkers comprise one ormore modifications that increase their binding affinity for one anotherand/or introduce a non-naturally occurring disulfide bond. In someembodiments, the first and second peptide linkers comprise aknob-into-hole modification (see, for example, Carter P. J ImmunolMethods. 248:7-15, 2001). In some embodiments, the first and secondpeptide linkers are modified by electrostatic steering to enhance theirassociation with one another (see, for example, WO2006106905 andGunasekaran K, et al. J Biol Chem. 285:19637-46, 2010). In someembodiments, the Fv-like antigen-binding module is human, humanized,chimeric, semi-synthetic, or fully synthetic.

In some embodiments, the antibody moiety (e.g., Fab-like antigen-bindingmodule or Fv-like antigen-binding module) is semi-synthetic, comprisingfully human sequences and one or more synthetic regions. In someembodiments, the antibody moiety is semi-synthetic, comprising a fullyhuman V_(L) and a semi-synthetic V_(H) comprising fully human FR1,HC-CDR1, FR2, HC-CDR2, FR3, and FR4 regions and a synthetic HC-CDR3. Insome embodiments, the semi-synthetic V_(H) comprises a fully syntheticHC-CDR3 having a sequence from about 5 to about 25 (such as about any of5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25) amino acids in length. In some embodiments, thesemi-synthetic V_(H) or the synthetic HC-CDR3 is obtained from asemi-synthetic library (such as a semi-synthetic human library)comprising fully synthetic HC-CDR3 regions having a sequence from about5 to about 25 (such as about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acids in length,wherein each amino acid in the sequence is randomly selected from thestandard human amino acids, minus cysteine. In some embodiments, thesynthetic HC-CDR3 is from about 10 to about 19 (such as about any of 10,11, 12, 13, 14, 15, 16, 17, 18 or 19) amino acids in length. In someembodiments, the antibody moiety is semi-synthetic, comprising asemi-synthetic V_(L) and a semi-synthetic V_(H). In some embodiments,the antibody moiety is fully-synthetic, comprising antibodies with fixedhuman V_(H)/V_(L) framework pairings, but randomized and syntheticsequences for all 6 CDRs of both heavy and light chains.

The antibody moiety (e.g., Fab-like antigen-binding module or Fv-likeantigen-binding module) in some embodiments comprises specific CDRsequences derived from one or more antibody moieties (such as amonoclonal antibody) or certain variants of such sequences comprisingone or more amino acid substitutions. In some embodiments, the aminoacid substitutions in the variant sequences do not substantially reducethe ability of the antibody moiety to bind the target antigen.Alterations that substantially improve target antigen binding affinityor affect some other property, such as specificity and/orcross-reactivity with related variants of the target antigen, are alsocontemplated.

The TCRM comprises a) a first polypeptide chain comprising a first Tcell receptor domain (TCRD) comprising a first transmembrane domain andb) a second polypeptide chain comprising a second TCRD comprising asecond transmembrane domain. In some embodiments, the firsttransmembrane domain is the transmembrane domain of a first TCR subunitand/or the second transmembrane domain is the transmembrane domain of asecond TCR subunit. In some embodiments, the first TCR subunit is a TCRα chain (e.g., GenBank Accession No: CCI73895), and the second TCRsubunit is a TCR β chain (e.g., GenBank Accession No: CCI73893). In someembodiments, the first TCR subunit is a TCR β chain, and the second TCRsubunit is a TCR α chain. In some embodiments, the first TCR subunit isa TCR γ chain (e.g., GenBank Accession No: AGE91788), and the second TCRsubunit is a TCR δ chain (e.g., GenBank Accession No: AAQ57272). In someembodiments, the first TCR subunit is a TCR δ chain, and the second TCRsubunit is a TCR γ chain. In some embodiments, the first and/or secondtransmembrane domains comprise (such as consist of), individually, atransmembrane domain contained in one of the amino acid sequences of SEQID NOs: 77-80. In some embodiments, the first and/or secondtransmembrane domains comprise (such as consist of), individually, anyone of the amino acid sequences of SEQ ID NOs: 1-4. In some embodiments,the first TCRD further comprises a first connecting peptideamino-terminal to the transmembrane domain and/or the second TCRDfurther comprises a second connecting peptide amino-terminal to thetransmembrane domain. In some embodiments, the first connecting peptidecomprises all or a portion of the connecting peptide of the first TCRsubunit and/or the second connecting peptide comprises all or a portionof the connecting peptide of the second TCR subunit. In someembodiments, the first transmembrane domain and the first connectingpeptide are derived from different TCR subunits and/or the secondtransmembrane domain and the second connecting peptide are derived fromdifferent TCR subunits. In some embodiments, the first and/or secondconnecting peptides comprise (such as consist of), individually, aconnecting peptide or fragment thereof contained in one of the aminoacid sequences of SEQ ID NOs: 77-80. In some embodiments, the firstand/or second connecting peptides comprise (such as consist of),individually, any one of the amino acid sequences of SEQ ID NOs: 5-12.In some embodiments, the first TCRD further comprises a first TCRintracellular domain carboxy-terminal to the first transmembrane domainand/or the second TCRD further comprises a second TCR intracellulardomain carboxy-terminal to the second transmembrane domain. In someembodiments, the first TCR intracellular domain comprises all or aportion of the intracellular domain of the first TCR subunit and/or thesecond TCR intracellular domain comprises all or a portion of theintracellular domain of the second TCR subunit. In some embodiments, thefirst and/or second TCR intracellular domains comprise, individually,all or a portion of an intracellular domain contained in any one of theamino acid sequences of SEQ ID NOs: 77-80. In some embodiments, thefirst and/or second TCR intracellular domains comprise, individually,any one of the amino acid sequences of SEQ ID NOs: 13-14. In someembodiments, the first TCRD is a fragment of the first TCR subunitand/or the second TCRD is a fragment of the second TCR chain. In someembodiments, the first and second polypeptide chains are linked, such asby a covalent linkage (e.g., peptide or other chemical linkage) ornon-covalent linkage. In some embodiments, the first and second TCRDsare linked by a disulfide bond. In some embodiments, the first andsecond TCRDs are linked by a disulfide bond between a residue in thefirst connecting peptide and a residue in the second connecting peptide.In some embodiments, the TCRM is capable of recruiting at least oneTCR-associated signaling module selected from the group consisting ofCD3δε, CD3γε, and ζζ. In some embodiments, the TCRM is capable ofrecruiting each of CD3δε, CD3γε, and ζζ to form an octameric abTCR-CD3complex (i.e., promotes abTCR-CD3 complex formation).

In some embodiments, the abTCR is a molecule comprising a fusion of thefirst polypeptide chain of the antibody moiety (e.g., Fab-likeantigen-binding module or Fv-like antigen-binding module) amino-terminalto the first polypeptide chain of the TCRM, thereby forming a firstpolypeptide chain of the abTCR, and a fusion of the second polypeptidechain of the antibody moiety amino-terminal to the second polypeptidechain of the TCRM, thereby forming a second polypeptide chain of theabTCR. In some embodiments, the abTCR further comprises a first peptidelinker between the first polypeptide chain of the antibody moiety andthe first polypeptide chain of the TCRM and/or a second peptide linkerbetween the second polypeptide chain of the antibody moiety and thesecond polypeptide chain of the TCRM. In some embodiments, the firstand/or second peptide linker is between about 5 to about 70 (such asabout any of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70,including any ranges between these values) amino acids in length. Insome embodiments, the first polypeptide chain of the abTCR furthercomprises a first signal peptide amino-terminal to the firstantigen-binding domain and/or the second polypeptide chain of the abTCRfurther comprises a second signal peptide amino-terminal to the secondantigen-binding domain. In some embodiments, the first and/or secondsignal peptides comprise (such as consist of) the amino acid sequence ofSEQ ID NO: 49. In some embodiments, the first polypeptide chain of theabTCR further comprises a first accessory intracellular domaincarboxy-terminal to the first transmembrane domain and/or the secondpolypeptide chain of the abTCR further comprises a second accessoryintracellular domain carboxy-terminal to the second transmembranedomain. In some embodiments, the first and/or second accessoryintracellular domains comprise a TCR costimulatory domain. In someembodiments, the TCR costimulatory domain comprises all or a portion ofthe amino acid sequence of SEQ ID NO: 70 or 71. In some embodiments, thefirst and/or second accessory intracellular domains comprise an epitopetag. In some embodiments, the epitope tag comprises any one of the aminoacid sequences of SEQ ID NOs: 50-52. In some embodiments, the first andsecond polypeptide chains of the abTCR are linked, such as by a covalentlinkage (e.g., peptide or other chemical linkage) or non-covalentlinkage. In some embodiments, the abTCR is a heterodimer.

In some embodiments, the target antigen is a cell surface antigen. Insome embodiments, the cell surface antigen is selected from the groupconsisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigenexpressed in a diseased cell. In some embodiments, the target antigen isa complex comprising a peptide and an MHC protein. Peptide/MHC complexesinclude, for example, a surface-presented complex comprising a peptidederived from a disease-associated antigen expressed in a diseased celland an MHC protein. In some embodiments, the full-lengthdisease-associated antigen is not normally expressed on the surface ofthe diseased cell (e.g., the disease-associated antigen is anintracellular or secreted protein). In some embodiments, the disease iscancer and the disease-associated antigen is a tumor-associated antigenexpressed in a cancer cell. In some embodiments, the tumor-associatedantigen is an oncoprotein. In some embodiments, the oncoprotein is theresult of a mutation in a proto-oncogene, and the oncoprotein comprisesa neoepitope comprising the mutation. For example, in some embodiments,the target antigen is a cell surface tumor-associated antigen (e.g., anoncoprotein comprising a neoepitope). In some embodiments, the targetantigen is a complex comprising a peptide derived from atumor-associated antigen (e.g., an oncoprotein comprising a neoepitope)not normally expressed on the surface of a cancer cell (e.g., anintracellular or secreted tumor-associated antigen) and an MHC protein.In some embodiments, the disease is viral infection and thedisease-associated antigen is a virus-associated antigen expressed in aninfected cell. For example, in some embodiments, the target antigen is acell surface virus-associated antigen. In some embodiments, the targetantigen is a complex comprising a peptide derived from avirus-associated antigen not normally expressed on the surface of avirus-infected cell (e.g., an intracellular or secreted virus-associatedantigen) and an MHC protein. In some embodiments, the abTCR constructbinds the target antigen with a K_(d) between about 0.1 pM to about 500nM (such as about any of 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1nM, 10 nM, 50 nM, 100 nM, or 500 nM, including any ranges between thesevalues).

In some embodiments, the abTCR comprises an antibody moiety (e.g.,Fab-like antigen-binding module or Fv-like antigen-binding module) thatspecifically binds to a cell surface antigen, wherein the cell surfaceantigen is CD19, ROR1, ROR2, BCMA, GPRC5D, or FCRL5. Specific binding toa full antigen, e.g., a cell surface antigen, is sometimes referred toas “non-MHC-restricted binding”.

In some embodiments, the abTCR comprises an antibody moiety (e.g.,Fab-like antigen-binding module or Fv-like antigen-binding module) thatspecifically binds to a complex comprising a peptide and an MHC protein,wherein the peptide is derived from a protein selected from the groupconsisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1,and PSA. Specific binding to a complex comprising a peptide and an MHCprotein is sometimes referred to as “MHC-restricted binding”.

In some embodiments, the abTCR comprises an antibody moiety (e.g.,Fab-like antigen-binding module or Fv-like antigen-binding module) thatspecifically binds to a complex comprising a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC class I protein, wherein the MHCclass I protein is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. In someembodiments, the MHC class I protein is HLA-A, HLA-B, or HLA-C. In someembodiments, the MHC class I protein is HLA-A. In some embodiments, theMHC class I protein is HLA-B. In some embodiments, the MHC class Iprotein is HLA-C. In some embodiments, the MHC class I protein isHLA-A01, HLA-A02, HLA-A03, HLA-A09, HLA-A10, HLA-A11, HLA-A19, HLA-A23,HLA-A24, HLA-A25, HLA-A26, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32,HLA-A33, HLA-A34, HLA-A36, HLA-A43, HLA-A66, HLA-A68, HLA-A69, HLA-A74,or HLA-A80. In some embodiments, the MHC class I protein is HLA-A02. Insome embodiments, the MHC class I protein is any one of HLA-A*02:01-555,such as HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05,HLA-A*02:06, HLA-A*02:07, HLA-A*02:08, HLA-A*02:09, HLA-A*02:10,HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:15,HLA-A*02:16, HLA-A*02:17, HLA-A*02:18, HLA-A*02:19, HLA-A*02:20,HLA-A*02:21, HLA-A*02:22, or HLA-A*02:24. In some embodiments, the MHCclass I protein is HLA-A*02:01.

In some embodiments, the abTCR comprises an antibody moiety (e.g.,Fab-like antigen-binding module or Fv-like antigen-binding module) thatspecifically binds to a complex comprising a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC class II protein, wherein the MHCclass II protein is HLA-DP, HLA-DQ, or HLA-DR. In some embodiments, theMHC class II protein is HLA-DP. In some embodiments, the MHC class IIprotein is HLA-DQ. In some embodiments, the MHC class II protein isHLA-DR.

For example, in some embodiments, there is provided an abTCR (such as anisolated abTCR) comprising a) a Fab-like antigen-binding module thatspecifically binds to a target antigen, and b) a TCRM capable ofrecruiting at least one TCR-associated signaling module. In someembodiments, the Fab-like antigen-binding module comprises a V_(H)antibody domain, a C_(H)1 antibody domain, a V_(L) antibody domain, anda C_(L) antibody domain. In some embodiments, the C_(H)1 domain isderived from an IgG (e.g, IgG1, IgG2, IgG3, or IgG4) heavy chain,optionally human. In some embodiments, the C_(H)1 domain is a variantcomprising one or more modifications (e.g., amino acid substitutions,insertions, and/or deletions) compared to the sequence from which it isderived. In some embodiments, the C_(L) domain is derived from a kappaor lambda light chain, optionally human. In some embodiments, the C_(L)domain is a variant comprising one or more modifications (e.g., aminoacid substitutions, insertions, and/or deletions) compared to thesequence from which it is derived. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the TCRM comprises thetransmembrane domains of a TCR, such as an αβTCR or a γδTCR. In someembodiments, the TCRM further comprises the connecting peptides orfragments thereof of a TCR, such as an αβTCR or a γδTCR. In someembodiments, the transmembrane domains and the connecting peptides arederived from an αβTCR or a γδTCR. In some embodiments, the transmembranedomains are derived from an αβTCR and the connecting peptides arederived from a γδTCR, or the transmembrane domains are derived from aγδTCR and the connecting peptides are derived from an αβTCR. In someembodiments, the TCRM further comprises at least one portion of anextracellular domain of the TCR. In some embodiments, the TCRM furthercomprises at least one TCR intracellular domain comprising a sequencefrom an intracellular domain of the TCR. In some embodiments, the TCRMcomprises fragments of the TCR subunits. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga T cell costimulatory signaling sequence (such as from CD27, CD28,4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA,FLAG, or myc). In some embodiments, the abTCR further comprises a firstsignal peptide amino-terminal to the first antigen-binding domain and/ora second signal peptide amino-terminal to the second antigen-bindingdomain. In some embodiments, the abTCR further comprises at least onedisulfide bond. In some embodiments, the Fab-like antigen binding modulecomprises a disulfide bond and/or the TCRM comprises a disulfide bond.In some embodiments, the Fab-like antigen binding module comprises adisulfide bond between a residue in the C_(H)1 domain and a residue inthe C_(L) domain and/or the TCRM comprises a disulfide bond between aresidue in the first connecting peptide and a residue in the secondconnecting peptide. In some embodiments, the TCRM is capable ofrecruiting at least one TCR-associated signaling module selected fromthe group consisting of CD3δε, CD3γε, and ζζ. In some embodiments, theTCRM promotes abTCR-CD3 complex formation. In some embodiments, there isa peptide linker between the Fab-like antigen-binding module and theTCRM. In some embodiments, the target antigen is a cell surface antigen.In some embodiments, the cell surface antigen is selected from the groupconsisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01.

In some embodiments, there is provided an abTCR (such as an isolatedabTCR) comprising a) an Fv-like antigen-binding module that specificallybinds to a target antigen, and b) a TCRM capable of recruiting at leastone TCR-associated signaling module, wherein the target antigen is apeptide/MHC complex. In some embodiments, the Fv-like antigen-bindingmodule comprises a V_(H) antibody domain and a V_(L) antibody domain. Insome embodiments, there is a first peptide linker fused to theC-terminus of the V_(L) antibody domain and/or a second peptide linkerfused to the C-terminus of the V_(H) antibody domain. In someembodiments, the first and second peptide linkers are capable of bindingto one another. In some embodiments, the first and/or second peptidelinkers are derived from immunoglobulin heavy and/or light chainconstant regions. In some embodiments, the first and/or second peptidelinkers are derived from TCR subunit constant regions. For example, insome embodiments, the first and/or second peptide linkers are derivedfrom a) TCR α and β subunit constant domains; or b) TCR γ and δ subunitconstant domains. In some embodiments, the first and/or second peptidelinkers are synthetic. In some embodiments, the Fv-like antigen-bindingmodule is human, humanized, chimeric, semi-synthetic, or fullysynthetic. In some embodiments, the TCRM comprises the transmembranedomains of a TCR, such as an αβTCR or a γδTCR. In some embodiments, theTCRM further comprises the connecting peptides or fragments thereof of aTCR, such as an αβTCR or a γδTCR. In some embodiments, the transmembranedomains and the connecting peptides are derived from an αβTCR or aγδTCR. In some embodiments, the transmembrane domains are derived froman αβTCR and the connecting peptides are derived from a γδTCR, or thetransmembrane domains are derived from a γδTCR and the connectingpeptides are derived from an αβTCR. In some embodiments, the TCRMfurther comprises at least one portion of an extracellular domain of theTCR. In some embodiments, the TCRM further comprises at least one TCRintracellular domain comprising a sequence from an intracellular domainof the TCR. In some embodiments, the TCRM comprises fragments of the TCRsubunits. In some embodiments, the abTCR further comprises at least oneaccessory intracellular domain comprising a T cell costimulatorysignaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30,or CD40) and/or an epitope tag (such as HA, FLAG, or myc). In someembodiments, the abTCR further comprises a first signal peptideamino-terminal to the first antigen-binding domain and/or a secondsignal peptide amino-terminal to the second antigen-binding domain. Insome embodiments, the abTCR further comprises at least one disulfidebond. In some embodiments, the first and/or second peptide linkerscomprise a disulfide bond and/or the TCRM comprises a disulfide bond. Insome embodiments, the TCRM comprises a disulfide bond between a residuein the first connecting peptide and a residue in the second connectingpeptide. In some embodiments, the TCRM is capable of recruiting at leastone TCR-associated signaling module selected from the group consistingof CD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotesabTCR-CD3 complex formation. In some embodiments, the target antigenpeptide/MHC complex comprises a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC protein. In some embodiments, thepeptide/MHC complex comprises a peptide and an MHC protein, wherein thepeptide is derived from a protein selected from the group consisting ofWT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, and PSA. In someembodiments, the MHC protein is an MHC class I protein. In someembodiments, the MHC class I protein is HLA-A. In some embodiments, theHLA-A is HLA-A02. In some embodiments, the HLA-A02 is HLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a target antigen comprising a) a first polypeptide chaincomprising a first antigen-binding domain comprising V_(H) and C_(H)1antibody domains and a first TCRD comprising the transmembrane domain ofa first TCR subunit; and b) a second polypeptide chain comprising asecond antigen-binding domain comprising V_(L) and C_(L) antibodydomains and a second TCRD comprising the transmembrane domain of asecond TCR subunit, wherein the V_(H) and C_(H)1 domains of the firstantigen-binding domain and the V_(L) and C_(L) domains of the secondantigen-binding domain form a Fab-like antigen-binding module thatspecifically binds the target antigen, wherein the first TCRD and thesecond TCRD form a TCRM that is capable of recruiting at least oneTCR-associated signaling module. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the first TCR subunit is a TCR αchain, and the second TCR subunit is a TCR β chain. In some embodiments,the first TCR subunit is a TCR β chain, and the second TCR subunit is aTCR α chain. In some embodiments, the first TCR subunit is a TCR γchain, and the second TCR subunit is a TCR δ chain. In some embodiments,the first TCR subunit is a TCR δ chain, and the second TCR subunit is aTCR γ chain. In some embodiments, the first TCRD further comprises theconnecting peptide or a fragment thereof of the first TCR subunit and/orthe second TCRD further comprises the connecting peptide or a fragmentthereof of the second TCR subunit. In some embodiments, the first TCRDfurther comprises a portion of the extracellular domain of the first TCRsubunit and/or the second TCRD further comprises a portion of theextracellular domain of the second TCR subunit. In some embodiments, thefirst TCRD further comprises a first TCR intracellular domain and/or thesecond TCRD further comprises a second TCR intracellular domain. In someembodiments, the first TCR intracellular domain comprises a sequencefrom the intracellular domain of the first TCR subunit and/or the secondTCR intracellular domain comprises a sequence from the intracellulardomain of the second TCR subunit. In some embodiments, the first TCRD isa fragment of the first TCR subunit and/or the second TCRD is a fragmentof the second TCR chain. In some embodiments, the abTCR furthercomprises at least one accessory intracellular domain comprising a Tcell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the abTCR further comprises a first signalpeptide amino-terminal to the first antigen-binding domain and/or asecond signal peptide amino-terminal to the second antigen-bindingdomain. In some embodiments, the TCRM is capable of recruiting at leastone TCR-associated signaling module selected from the group consistingof CD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotesabTCR-CD3 complex formation. In some embodiments, there is a firstpeptide linker between the first antigen-binding domain and the firstTCRD and/or a second peptide linker between the second antigen-bindingdomain and the second TCRD. In some embodiments, the first and secondpolypeptide chains are linked, such as by a covalent linkage (e.g.,peptide or other chemical linkage) or non-covalent linkage. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between a residuein the C_(H)1 antibody domain in the first antigen-binding domain and aresidue in the C_(L) antibody domain in the second antigen-bindingdomain. In some embodiments, the C_(H)1 domain is derived from an IgG(e.g, IgG1, IgG2, IgG3, or IgG4) heavy chain, optionally human. In someembodiments, the C_(H)1 domain is a variant comprising one or moremodifications (e.g., amino acid substitutions, insertions, and/ordeletions) compared to the sequence from which it is derived. In someembodiments, the C_(L) domain is derived from a kappa or lambda lightchain, optionally human. In some embodiments, the C_(L) domain is avariant comprising one or more modifications (e.g., amino acidsubstitutions, insertions, and/or deletions) compared to the sequencefrom which it is derived. In some embodiments, the target antigen is acell surface antigen. In some embodiments, the cell surface antigen isselected from the group consisting of a protein, a carbohydrate, and alipid. In some embodiments, the cell surface antigen is adisease-associated antigen, such as a tumor-associated orvirally-encoded antigen. In some embodiments, the cell surface antigenis CD19, ROR1, ROR2, BCMA, GPRC5D, or FCRL5. In some embodiments, thetarget antigen is a surface-presented peptide/MHC complex. In someembodiments, the peptide/MHC complex comprises a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC protein. In some embodiments, thepeptide/MHC complex comprises a peptide and an MHC protein, wherein thepeptide is derived from a protein selected from the group consisting ofWT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, and PSA. In someembodiments, the MHC protein is an MHC class I protein. In someembodiments, the MHC class I protein is HLA-A. In some embodiments, theHLA-A is HLA-A02. In some embodiments, the HLA-A02 is HLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a target antigen comprising a) a first polypeptide chaincomprising a first antigen-binding domain comprising a V_(H) antibodydomain and a first TCRD comprising the transmembrane domain of a firstTCR subunit; and b) a second polypeptide chain comprising a secondantigen-binding domain comprising a V_(L) antibody domain and a secondTCRD comprising the transmembrane domain of a second TCR subunit,wherein the V_(H) domain of the first antigen-binding domain and theV_(L) domain of the second antigen-binding domain form an Fv-likeantigen-binding module that specifically binds the target antigen,wherein the first TCRD and the second TCRD form a TCRM that is capableof recruiting at least one TCR-associated signaling module, and whereinthe target antigen is a peptide/MHC complex. In some embodiments, theFv-like antigen-binding module is human, humanized, chimeric,semi-synthetic, or fully synthetic. In some embodiments, the first TCRsubunit is a TCR α chain, and the second TCR subunit is a TCR β chain.In some embodiments, the first TCR subunit is a TCR β chain, and thesecond TCR subunit is a TCR α chain. In some embodiments, the first TCRsubunit is a TCR γ chain, and the second TCR subunit is a TCR δ chain.In some embodiments, the first TCR subunit is a TCR δ chain, and thesecond TCR subunit is a TCR γ chain. In some embodiments, the first TCRDfurther comprises the connecting peptide or a fragment thereof of thefirst TCR subunit and/or the second TCRD further comprises theconnecting peptide or a fragment thereof of the second TCR subunit. Insome embodiments, the first TCRD further comprises a portion of theextracellular domain of the first TCR subunit and/or the second TCRDfurther comprises a portion of the extracellular domain of the secondTCR subunit. In some embodiments, the first TCRD further comprises afirst TCR intracellular domain and/or the second TCRD further comprisesa second TCR intracellular domain. In some embodiments, the first TCRintracellular domain comprises a sequence from the intracellular domainof the first TCR subunit and/or the second TCR intracellular domaincomprises a sequence from the intracellular domain of the second TCRsubunit. In some embodiments, the first TCRD is a fragment of the firstTCR subunit and/or the second TCRD is a fragment of the second TCRchain. In some embodiments, the abTCR further comprises at least oneaccessory intracellular domain comprising a T cell costimulatorysignaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30,or CD40) and/or an epitope tag (such as HA, FLAG, or myc). In someembodiments, the abTCR further comprises a first signal peptideamino-terminal to the first antigen-binding domain and/or a secondsignal peptide amino-terminal to the second antigen-binding domain. Insome embodiments, the TCRM is capable of recruiting at least oneTCR-associated signaling module selected from the group consisting ofCD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotes abTCR-CD3complex formation. In some embodiments, there is a first peptide linkerbetween the first antigen-binding domain and the first TCRD and/or asecond peptide linker between the second antigen-binding domain and thesecond TCRD. In some embodiments, the first and/or second peptidelinkers are derived from immunoglobulin heavy and/or light chainconstant regions. In some embodiments, the first and/or second peptidelinkers are derived from TCR subunit constant regions. For example, insome embodiments, the first and/or second peptide linkers are derivedfrom a) TCR α and β subunit constant domains; or b) TCR γ and δ subunitconstant domains. In some embodiments, the first and/or second peptidelinkers are synthetic. In some embodiments, the first and secondpolypeptide chains are linked, such as by a covalent linkage (e.g.,peptide or other chemical linkage) or non-covalent linkage. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between a residuein the first peptide linker and a residue in the second peptide linker.In some embodiments, the first and/or second peptide linker is a variantcomprising one or more modifications (e.g., amino acid substitutions,insertions, and/or deletions) compared to the sequence from which it isderived. In some embodiments, the first and/or second peptide linkerscomprise one or more modifications that do not substantially alter theirbinding affinities for one another. In some embodiments, the firstand/or second peptide linkers comprise one or more modifications thatincrease their binding affinities for one another and/or introduce anon-naturally occurring disulfide bond. In some embodiments, the targetantigen peptide/MHC complex comprises a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC protein. In some embodiments, thepeptide/MHC complex comprises a peptide and an MHC protein, wherein thepeptide is derived from a protein selected from the group consisting ofWT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, and PSA. In someembodiments, the MHC protein is an MHC class I protein. In someembodiments, the MHC class I protein is HLA-A. In some embodiments, theHLA-A is HLA-A02. In some embodiments, the HLA-A02 is HLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a target antigen comprising a) a first polypeptide chaincomprising a first antigen-binding domain comprising V_(H) and C_(H)1antibody domains and a first TCRD comprising the transmembrane domain ofa TCR α chain; and b) a second polypeptide chain comprising a secondantigen-binding domain comprising V_(L) and C_(L) antibody domains and asecond TCRD comprising the transmembrane domain of a TCR β chain,wherein the V_(H) and C_(H)1 domains of the first antigen-binding domainand the V_(L) and C_(L) domains of the second antigen-binding domainform a Fab-like antigen-binding module that specifically binds thetarget antigen, wherein the first TCRD and the second TCRD form a TCRMthat is capable of recruiting at least one TCR-associated signalingmodule. In some embodiments, the Fab-like antigen-binding module ishuman, humanized, chimeric, semi-synthetic, or fully synthetic. In someembodiments, the first TCRD further comprises the connecting peptide ora fragment thereof of the TCR α chain and/or the second TCRD furthercomprises the connecting peptide or a fragment thereof of the TCR βchain. In some embodiments, the first TCRD further comprises a portionof the extracellular domain of the TCR α chain and/or the second TCRDfurther comprises a portion of the extracellular domain of the TCR βchain. In some embodiments, the first TCRD further comprises a first TCRintracellular domain and/or the second TCRD further comprises a secondTCR intracellular domain. In some embodiments, the first TCRintracellular domain comprises a sequence from the intracellular domainof the TCR α chain and/or the second TCR intracellular domain comprisesa sequence from the intracellular domain of the TCR β chain. In someembodiments, the abTCR further comprises at least one accessoryintracellular domain comprising a T cell costimulatory signalingsequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD40)and/or an epitope tag (such as HA, FLAG, or myc). In some embodiments,the abTCR further comprises a first signal peptide amino-terminal to thefirst antigen-binding domain and/or a second signal peptideamino-terminal to the second antigen-binding domain. In someembodiments, the TCRM is capable of recruiting at least oneTCR-associated signaling module selected from the group consisting ofCD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotes abTCR-CD3complex formation. In some embodiments, there is a first peptide linkerbetween the first antigen-binding domain and the first TCRD and/or asecond peptide linker between the second antigen-binding domain and thesecond TCRD. In some embodiments, the first and second polypeptidechains are linked, such as by a covalent linkage (e.g., peptide or otherchemical linkage) or non-covalent linkage. In some embodiments, thefirst polypeptide chain and the second polypeptide chain are linked viaa) a disulfide bond between a residue in the connecting peptide of thefirst TCRD and a residue in the connecting peptide of the second TCRD;and/or b) a disulfide bond between a residue in the C_(H)1 antibodydomain in the first antigen-binding domain and a residue in the C_(L)antibody domain in the second antigen-binding domain. In someembodiments, the target antigen is a cell surface antigen. In someembodiments, the cell surface antigen is selected from the groupconsisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a target antigen comprising a) a first polypeptide chaincomprising a first antigen-binding domain comprising V_(H) and C_(H)1antibody domains and a first TCRD comprising the transmembrane domain ofa TCR β chain; and b) a second polypeptide chain comprising a secondantigen-binding domain comprising V_(L) and C_(L) antibody domains and asecond TCRD comprising the transmembrane domain of a TCR α chain,wherein the V_(H) and C_(H)1 domains of the first antigen-binding domainand the V_(L) and C_(L) domains of the second antigen-binding domainform a Fab-like antigen-binding module that specifically binds thetarget antigen, wherein the first TCRD and the second TCRD form a TCRMthat is capable of recruiting at least one TCR-associated signalingmodule. In some embodiments, the Fab-like antigen-binding module ishuman, humanized, chimeric, semi-synthetic, or fully synthetic. In someembodiments, the first TCRD further comprises the connecting peptide ora fragment thereof of the TCR β chain and/or the second TCRD furthercomprises the connecting peptide or a fragment thereof of the TCR αchain. In some embodiments, the first TCRD further comprises a portionof the extracellular domain of the TCR β chain and/or the second TCRDfurther comprises a portion of the extracellular domain of the TCR αchain. In some embodiments, the first TCRD further comprises a first TCRintracellular domain and/or the second TCRD further comprises a secondTCR intracellular domain. In some embodiments, the first TCRintracellular domain comprises a sequence from the intracellular domainof the TCR β chain and/or the second TCR intracellular domain comprisesa sequence from the intracellular domain of the TCR α chain. In someembodiments, the abTCR further comprises at least one accessoryintracellular domain comprising a T cell costimulatory signalingsequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD40)and/or an epitope tag (such as HA, FLAG, or myc). In some embodiments,the abTCR further comprises a first signal peptide amino-terminal to thefirst antigen-binding domain and/or a second signal peptideamino-terminal to the second antigen-binding domain. In someembodiments, the TCRM is capable of recruiting at least oneTCR-associated signaling module selected from the group consisting ofCD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotes abTCR-CD3complex formation. In some embodiments, there is a first peptide linkerbetween the first antigen-binding domain and the first TCRD and/or asecond peptide linker between the second antigen-binding domain and thesecond TCRD. In some embodiments, the first and second polypeptidechains are linked, such as by a covalent linkage (e.g., peptide or otherchemical linkage) or non-covalent linkage. In some embodiments, thefirst polypeptide chain and the second polypeptide chain are linked viaa) a disulfide bond between a residue in the connecting peptide of thefirst TCRD and a residue in the connecting peptide of the second TCRD;and/or b) a disulfide bond between a residue in the C_(H)1 antibodydomain in the first antigen-binding domain and a residue in the C_(L)antibody domain in the second antigen-binding domain. In someembodiments, the target antigen is a cell surface antigen. In someembodiments, the cell surface antigen is selected from the groupconsisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a target antigen comprising a) a first polypeptide chaincomprising a first antigen-binding domain comprising V_(H) and C_(H)1antibody domains and a first TCRD comprising the transmembrane domain ofa TCR γ chain; and b) a second polypeptide chain comprising a secondantigen-binding domain comprising V_(L) and C_(L) antibody domains and asecond TCRD comprising the transmembrane domain of a TCR δ chain,wherein the V_(H) and C_(H)1 domains of the first antigen-binding domainand the V_(L) and C_(L) domains of the second antigen-binding domainform a Fab-like antigen-binding module that specifically binds thetarget antigen, wherein the first TCRD and the second TCRD form a TCRMthat is capable of recruiting at least one TCR-associated signalingmodule. In some embodiments, the Fab-like antigen-binding module ishuman, humanized, chimeric, semi-synthetic, or fully synthetic. In someembodiments, the first TCRD further comprises the connecting peptide ora fragment thereof of the TCR γ chain and/or the second TCRD furthercomprises the connecting peptide or a fragment thereof of the TCR δchain. In some embodiments, the first TCRD further comprises a portionof the extracellular domain of the TCR γ chain and/or the second TCRDfurther comprises a portion of the extracellular domain of the TCR δchain. In some embodiments, the first TCRD further comprises a first TCRintracellular domain and/or the second TCRD further comprises a secondTCR intracellular domain. In some embodiments, the first TCRintracellular domain comprises a sequence from the intracellular domainof the TCR γ chain and/or the second TCR intracellular domain comprisesa sequence from the intracellular domain of the TCR δ chain. In someembodiments, the abTCR further comprises at least one accessoryintracellular domain comprising a T cell costimulatory signalingsequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD40)and/or an epitope tag (such as HA, FLAG, or myc). In some embodiments,the abTCR further comprises a first signal peptide amino-terminal to thefirst antigen-binding domain and/or a second signal peptideamino-terminal to the second antigen-binding domain. In someembodiments, the TCRM is capable of recruiting at least oneTCR-associated signaling module selected from the group consisting ofCD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotes abTCR-CD3complex formation. In some embodiments, there is a first peptide linkerbetween the first antigen-binding domain and the first TCRD and/or asecond peptide linker between the second antigen-binding domain and thesecond TCRD. In some embodiments, the first and second polypeptidechains are linked, such as by a covalent linkage (e.g., peptide or otherchemical linkage) or non-covalent linkage. In some embodiments, thefirst polypeptide chain and the second polypeptide chain are linked viaa) a disulfide bond between a residue in the connecting peptide of thefirst TCRD and a residue in the connecting peptide of the second TCRD;and/or b) a disulfide bond between a residue in the C_(H)1 antibodydomain in the first antigen-binding domain and a residue in the C_(L)antibody domain in the second antigen-binding domain. In someembodiments, the target antigen is a cell surface antigen. In someembodiments, the cell surface antigen is selected from the groupconsisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a target antigen comprising a) a first polypeptide chaincomprising a first antigen-binding domain comprising V_(H) and C_(H)1antibody domains and a first TCRD comprising the transmembrane domain ofa TCR δ chain; and b) a second polypeptide chain comprising a secondantigen-binding domain comprising V_(L) and C_(L) antibody domains and asecond TCRD comprising the transmembrane domain of a TCR γ chain,wherein the V_(H) and C_(H)1 domains of the first antigen-binding domainand the V_(L) and C_(L) domains of the second antigen-binding domainform a Fab-like antigen-binding module that specifically binds thetarget antigen, wherein the first TCRD and the second TCRD form a TCRMthat is capable of recruiting at least one TCR-associated signalingmodule. In some embodiments, the Fab-like antigen-binding module ishuman, humanized, chimeric, semi-synthetic, or fully synthetic. In someembodiments, the first TCRD further comprises the connecting peptide ora fragment thereof of the TCR δ chain and/or the second TCRD furthercomprises the connecting peptide or a fragment thereof of the TCR γchain. In some embodiments, the first TCRD further comprises a portionof the extracellular domain of the TCR δ chain and/or the second TCRDfurther comprises a portion of the extracellular domain of the TCR γchain. In some embodiments, the first TCRD further comprises a first TCRintracellular domain and/or the second TCRD further comprises a secondTCR intracellular domain. In some embodiments, the first TCRintracellular domain comprises a sequence from the intracellular domainof the TCR δ chain and/or the second TCR intracellular domain comprisesa sequence from the intracellular domain of the TCR γ chain. In someembodiments, the abTCR further comprises at least one accessoryintracellular domain comprising a T cell costimulatory signalingsequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD40)and/or an epitope tag (such as HA, FLAG, or myc). In some embodiments,the abTCR further comprises a first signal peptide amino-terminal to thefirst antigen-binding domain and/or a second signal peptideamino-terminal to the second antigen-binding domain. In someembodiments, the TCRM is capable of recruiting at least oneTCR-associated signaling module selected from the group consisting ofCD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotes abTCR-CD3complex formation. In some embodiments, there is a first peptide linkerbetween the first antigen-binding domain and the first TCRD and/or asecond peptide linker between the second antigen-binding domain and thesecond TCRD. In some embodiments, the first and second polypeptidechains are linked, such as by a covalent linkage (e.g., peptide or otherchemical linkage) or non-covalent linkage. In some embodiments, thefirst polypeptide chain and the second polypeptide chain are linked viaa) a disulfide bond between a residue in the connecting peptide of thefirst TCRD and a residue in the connecting peptide of the second TCRD;and/or b) a disulfide bond between a residue in the C_(H)1 antibodydomain in the first antigen-binding domain and a residue in the C_(L)antibody domain in the second antigen-binding domain. In someembodiments, the target antigen is a cell surface antigen. In someembodiments, the cell surface antigen is selected from the groupconsisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a target antigen comprising a) a first polypeptide chaincomprising a first antigen-binding domain comprising V_(H) and C_(H)1antibody domains and a first TCRD comprising a transmembrane domaincomprising (such as consisting of) the amino acid sequence of any one ofSEQ ID NOs: 1-4; and b) a second polypeptide chain comprising a secondantigen-binding domain comprising V_(L) and C_(L) antibody domains and asecond TCRD comprising a transmembrane domain comprising (such asconsisting of) the amino acid sequence of any one of SEQ ID NOs: 1-4,wherein the V_(H) and C_(H)1 domains of the first antigen-binding domainand the V_(L) and C_(L) domains of the second antigen-binding domainform a Fab-like antigen-binding module that specifically binds thetarget antigen, wherein the first TCRD and the second TCRD form a TCRMthat is capable of recruiting at least one TCR-associated signalingmodule. In some embodiments, the Fab-like antigen-binding module ishuman, humanized, chimeric, semi-synthetic, or fully synthetic. In someembodiments, the first TCRD further comprises a first connecting peptideor fragment thereof of a first TCR subunit and/or the second TCRDfurther comprises a second connecting peptide or fragment thereof of asecond TCR subunit, wherein the first and/or second connecting peptidescomprise (such as consist of) the amino acid sequence of any one of SEQID NOs: 5-12. In some embodiments, the first TCRD further comprises afirst TCR intracellular domain and/or the second TCRD further comprisesa second TCR intracellular domain, wherein the first and/or second TCRintracellular domains comprise (such as consist of) the amino sequenceof any one of SEQ ID NOs: 13-14. In some embodiments, the abTCR furthercomprises at least one accessory intracellular domain comprising a) atleast one T cell costimulatory signaling sequence comprising (such asconsisting of) the amino acid sequence of SEQ ID NO: 70 or 71; and/or b)an epitope tag comprising (such as consisting of) the amino acidsequence of any one of SEQ ID NOs: 50-52. In some embodiments, the abTCRfurther comprises a first signal peptide amino-terminal to the firstantigen-binding domain and/or a second signal peptide amino-terminal tothe second antigen-binding domain, wherein the first and/or secondsignal peptides comprise the amino acid sequence of SEQ ID NO: 49. Insome embodiments, the TCRM is capable of recruiting at least oneTCR-associated signaling module selected from the group consisting ofCD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotes abTCR-CD3complex formation. In some embodiments, there is a first peptide linkerbetween the first antigen-binding domain and the first TCRD and/or asecond peptide linker between the second antigen-binding domain and thesecond TCRD. In some embodiments, the first and second polypeptidechains are linked, such as by a covalent linkage (e.g., peptide or otherchemical linkage) or non-covalent linkage. In some embodiments, thefirst polypeptide chain and the second polypeptide chain are linked viaa) a disulfide bond between a residue in the connecting peptide of thefirst TCRD and a residue in the connecting peptide of the second TCRD;and/or b) a disulfide bond between a residue in the C_(H)1 antibodydomain in the first antigen-binding domain and a residue in the C_(L)antibody domain in the second antigen-binding domain. In someembodiments, the target antigen is a cell surface antigen. In someembodiments, the cell surface antigen is selected from the groupconsisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a target antigen comprising a) a first polypeptide chaincomprising a first antigen-binding domain comprising a V_(H) antibodydomain and a first TCRD comprising a transmembrane domain comprising(such as consisting of) the amino acid sequence of any one of SEQ IDNOs: 1-4; and b) a second polypeptide chain comprising a secondantigen-binding domain comprising a V_(L) antibody domain and a secondTCRD comprising a transmembrane domain comprising (such as consistingof) the amino acid sequence of any one of SEQ ID NOs: 1-4, wherein theV_(H) domain of the first antigen-binding domain and the V_(L) domain ofthe second antigen-binding domain form an Fv-like antigen-binding modulethat specifically binds the target antigen, wherein the first TCRD andthe second TCRD form a TCRM that is capable of recruiting at least oneTCR-associated signaling module, and wherein the target antigen is apeptide/MHC complex. In some embodiments, the Fv-like antigen-bindingmodule is human, humanized, chimeric, semi-synthetic, or fullysynthetic. In some embodiments, the first TCRD further comprises a firstconnecting peptide or fragment thereof of a first TCR subunit and/or thesecond TCRD further comprises a second connecting peptide or fragmentthereof of a second TCR subunit, wherein the first and/or secondconnecting peptides comprise (such as consist of) the amino acidsequence of any one of SEQ ID NOs: 5-12. In some embodiments, the firstTCRD further comprises a first TCR intracellular domain and/or thesecond TCRD further comprises a second TCR intracellular domain, whereinthe first and/or second TCR intracellular domains comprise (such asconsist of) the amino sequence of any one of SEQ ID NOs: 13-14. In someembodiments, the abTCR further comprises at least one accessoryintracellular domain comprising a) at least one T cell costimulatorysignaling sequence comprising (such as consisting of) the amino acidsequence of SEQ ID NO: 70 or 71; and/or b) an epitope tag comprising(such as consisting of) the amino acid sequence of any one of SEQ IDNOs: 50-52. In some embodiments, the abTCR further comprises a firstsignal peptide amino-terminal to the first antigen-binding domain and/ora second signal peptide amino-terminal to the second antigen-bindingdomain, wherein the first and/or second signal peptides comprise theamino acid sequence of SEQ ID NO: 49. In some embodiments, the TCRM iscapable of recruiting at least one TCR-associated signaling moduleselected from the group consisting of CD3δε, CD3γε, and ζζ. In someembodiments, the TCRM promotes abTCR-CD3 complex formation. In someembodiments, there is a first peptide linker between the firstantigen-binding domain and the first TCRD and/or a second peptide linkerbetween the second antigen-binding domain and the second TCRD. In someembodiments, the first and second peptide linkers are capable of bindingto one another. In some embodiments, the first and/or second peptidelinkers are derived from immunoglobulin heavy and/or light chainconstant regions. In some embodiments, the first and/or second peptidelinkers are derived from TCR subunit constant regions. For example, insome embodiments, the first and/or second peptide linkers are derivedfrom a) TCR α and β subunit constant domains; or b) TCR γ and δ subunitconstant domains. In some embodiments, the first and/or second peptidelinkers are synthetic. In some embodiments, the first and secondpolypeptide chains are linked, such as by a covalent linkage (e.g.,peptide or other chemical linkage) or non-covalent linkage. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between a residuein the first peptide linker and a residue in the second peptide linker.In some embodiments, the first and/or second peptide linker is a variantcomprising one or more modifications (e.g., amino acid substitutions,insertions, and/or deletions) compared to the sequence from which it isderived. In some embodiments, the first and/or second peptide linkerscomprise one or more modifications that do not substantially alter theirbinding affinities for one another. In some embodiments, the firstand/or second peptide linkers comprise one or more modifications thatincrease their binding affinities for one another and/or introduce anon-naturally occurring disulfide bond. In some embodiments, the targetantigen peptide/MHC complex comprises a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC protein. In some embodiments, thepeptide/MHC complex comprises a peptide and an MHC protein, wherein thepeptide is derived from a protein selected from the group consisting ofWT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, and PSA. In someembodiments, the MHC protein is an MHC class I protein. In someembodiments, the MHC class I protein is HLA-A. In some embodiments, theHLA-A is HLA-A02. In some embodiments, the HLA-A02 is HLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a target antigen comprising a) a first polypeptide chaincomprising, in order from amino terminus to carboxy terminus, a firstantigen-binding domain comprising V_(H) and C_(H)1 antibody domains, anda first TCRD comprising a connecting peptide comprising (such asconsisting of) the amino acid sequence of any one of SEQ ID NOs: 5-12and a transmembrane domain comprising (such as consisting of) the aminoacid sequence of any one of SEQ ID NOs: 1-4; and b) a second polypeptidechain comprising, in order from amino terminus to carboxy terminus, asecond antigen-binding domain comprising V_(L) and C_(L) antibodydomains and a second TCRD comprising a connecting peptide comprising(such as consisting of) the amino acid sequence of any one of SEQ IDNOs: 5-12 and a transmembrane domain comprising (such as consisting of)the amino acid sequence of any one of SEQ ID NOs: 1-4; wherein the V_(H)and C_(H)1 domains of the first antigen-binding domain and the V_(L) andC_(L) domains of the second antigen-binding domain form a Fab-likeantigen-binding module that specifically binds the target antigen,wherein the first TCRD and the second TCRD form a TCRM that is capableof recruiting at least one TCR-associated signaling module. In someembodiments, the Fab-like antigen-binding module is human, humanized,chimeric, semi-synthetic, or fully synthetic. In some embodiments, thefirst TCRD further comprises a first TCR intracellular domain and/or thesecond TCRD further comprises a second TCR intracellular domain, whereinthe first and/or second TCR intracellular domains comprise (such asconsist of) the amino sequence of any one of SEQ ID NOs: 13-14. In someembodiments, the abTCR further comprises at least one accessoryintracellular domain comprising at least one T cell costimulatorysignaling sequence comprising (such as consisting of) the amino acidsequence of SEQ ID NO: 70 or 71; and/or b) an epitope tag comprising(such as consisting of) the amino acid sequence of any one of SEQ IDNOs: 50-52. In some embodiments, the abTCR further comprises a firstsignal peptide amino-terminal to the first antigen-binding domain and/ora second signal peptide amino-terminal to the second antigen-bindingdomain, wherein the first and/or second signal peptides comprise theamino acid sequence of SEQ ID NO: 49. In some embodiments, the TCRM iscapable of recruiting at least one TCR-associated signaling moduleselected from the group consisting of CD3δε, CD3γε, and ζζ. In someembodiments, the TCRM promotes abTCR-CD3 complex formation. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between a residuein the C_(H)1 antibody domain in the first antigen-binding domain and aresidue in the C_(L) antibody domain in the second antigen-bindingdomain. In some embodiments, the target antigen is a cell surfaceantigen. In some embodiments, the cell surface antigen is selected fromthe group consisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a target antigen comprising a) a first polypeptide chaincomprising, in order from amino terminus to carboxy terminus, a firstantigen-binding domain comprising a V_(H) antibody domain, and a firstTCRD comprising a connecting peptide comprising (such as consisting of)the amino acid sequence of any one of SEQ ID NOs: 5-12 and atransmembrane domain comprising (such as consisting of) the amino acidsequence of any one of SEQ ID NOs: 1-4; and b) a second polypeptidechain comprising, in order from amino terminus to carboxy terminus, asecond antigen-binding domain comprising a V_(L) antibody domain, and asecond TCRD comprising a connecting peptide comprising (such asconsisting of) the amino acid sequence of any one of SEQ ID NOs: 5-12and a transmembrane domain comprising (such as consisting of) the aminoacid sequence of any one of SEQ ID NOs: 1-4, wherein the V_(H) domain ofthe first antigen-binding domain and the V_(L) domain of the secondantigen-binding domain form an Fv-like antigen-binding module thatspecifically binds the target antigen, wherein the first TCRD and thesecond TCRD form a TCRM that is capable of recruiting at least oneTCR-associated signaling module, and wherein the target antigen is apeptide/MHC complex. In some embodiments, the Fv-like antigen-bindingmodule is human, humanized, chimeric, semi-synthetic, or fullysynthetic. In some embodiments, the first TCRD further comprises a firstconnecting peptide or fragment thereof of a first TCR subunit and/or thesecond TCRD further comprises a second connecting peptide or fragmentthereof of a second TCR subunit, wherein the first and/or secondconnecting peptides comprise (such as consist of) the amino acidsequence of any one of SEQ ID NOs: 5-12. In some embodiments, the firstTCRD further comprises a first TCR intracellular domain and/or thesecond TCRD further comprises a second TCR intracellular domain, whereinthe first and/or second TCR intracellular domains comprise (such asconsist of) the amino sequence of any one of SEQ ID NOs: 13-14. In someembodiments, the abTCR further comprises at least one accessoryintracellular domain comprising a) at least one T cell costimulatorysignaling sequence comprising (such as consisting of) the amino acidsequence of SEQ ID NO: 70 or 71; and/or b) an epitope tag comprising(such as consisting of) the amino acid sequence of any one of SEQ IDNOs: 50-52. In some embodiments, the abTCR further comprises a firstsignal peptide amino-terminal to the first antigen-binding domain and/ora second signal peptide amino-terminal to the second antigen-bindingdomain, wherein the first and/or second signal peptides comprise theamino acid sequence of SEQ ID NO: 49. In some embodiments, the TCRM iscapable of recruiting at least one TCR-associated signaling moduleselected from the group consisting of CD3δε, CD3γε, and ζζ. In someembodiments, the TCRM promotes abTCR-CD3 complex formation. In someembodiments, there is a first peptide linker between the firstantigen-binding domain and the first TCRD and/or a second peptide linkerbetween the second antigen-binding domain and the second TCRD. In someembodiments, the first and second peptide linkers are capable of bindingto one another. In some embodiments, the first and/or second peptidelinkers are derived from immunoglobulin heavy and/or light chainconstant regions. In some embodiments, the first and/or second peptidelinkers are derived from TCR subunit constant regions. For example, insome embodiments, the first and/or second peptide linkers are derivedfrom a) TCR α and β subunit constant domains; or b) TCR γ and δ subunitconstant domains. In some embodiments, the first and/or second peptidelinkers are synthetic. In some embodiments, the first and secondpolypeptide chains are linked, such as by a covalent linkage (e.g.,peptide or other chemical linkage) or non-covalent linkage. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between a residuein the first peptide linker and a residue in the second peptide linker.In some embodiments, the first and/or second peptide linker is a variantcomprising one or more modifications (e.g., amino acid substitutions,insertions, and/or deletions) compared to the sequence from which it isderived. In some embodiments, the first and/or second peptide linkerscomprise one or more modifications that do not substantially alter theirbinding affinities for one another. In some embodiments, the firstand/or second peptide linkers comprise one or more modifications thatincrease their binding affinities for one another and/or introduce anon-naturally occurring disulfide bond. In some embodiments, the targetantigen peptide/MHC complex comprises a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC protein. In some embodiments, thepeptide/MHC complex comprises a peptide and an MHC protein, wherein thepeptide is derived from a protein selected from the group consisting ofWT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, and PSA. In someembodiments, the MHC protein is an MHC class I protein. In someembodiments, the MHC class I protein is HLA-A. In some embodiments, theHLA-A is HLA-A02. In some embodiments, the HLA-A02 is HLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a target antigen comprising a) a first polypeptide chaincomprising, in order from amino terminus to carboxy terminus, a firstantigen-binding domain and a first TCRD comprising the amino acidsequence of SEQ ID NO: 15; and b) a second polypeptide chain comprising,in order from amino terminus to carboxy terminus, a secondantigen-binding domain and a second TCRD comprising the amino acidsequence of SEQ ID NO: 16; wherein the first antigen-binding domain andthe second antigen-binding domain form a Fab-like antigen-binding modulethat specifically binds the target antigen, wherein the first TCRD andthe second TCRD form a TCRM that is capable of recruiting at least oneTCR-associated signaling module. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the abTCR further comprises atleast one accessory intracellular domain comprising a) at least one Tcell costimulatory signaling sequence comprising (such as consisting of)the amino acid sequence of SEQ ID NO: 70 or 71; and/or b) an epitope tagcomprising (such as consisting of) the amino acid sequence of any one ofSEQ ID NOs: 50-52. In some embodiments, the abTCR further comprises afirst signal peptide amino-terminal to the first antigen-binding domainand/or a second signal peptide amino-terminal to the secondantigen-binding domain, wherein the first and/or second signal peptidescomprise the amino acid sequence of SEQ ID NO: 49. In some embodiments,the TCRM is capable of recruiting at least one TCR-associated signalingmodule selected from the group consisting of CD3δε, CD3γε, and ζζ. Insome embodiments, the TCRM promotes abTCR-CD3 complex formation. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between residuesin the C_(H)1 and C_(L) antibody domains in the Fab-like antigen-bindingmodule. In some embodiments, the target antigen is a cell surfaceantigen. In some embodiments, the cell surface antigen is selected fromthe group consisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a target antigen comprising a) a first polypeptide chaincomprising, in order from amino terminus to carboxy terminus, a firstantigen-binding domain and a first TCRD comprising the amino acidsequence of SEQ ID NO: 17; and b) a second polypeptide chain comprising,in order from amino terminus to carboxy terminus, a secondantigen-binding domain and a second TCRD comprising the amino acidsequence of SEQ ID NO: 18; wherein the first antigen-binding domain andthe second antigen-binding domain form a Fab-like antigen-binding modulethat specifically binds the target antigen, wherein the first TCRD andthe second TCRD form a TCRM that is capable of recruiting at least oneTCR-associated signaling module. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the abTCR further comprises atleast one accessory intracellular domain comprising a) at least one Tcell costimulatory signaling sequence comprising (such as consisting of)the amino acid sequence of SEQ ID NO: 70 or 71; and/or b) an epitope tagcomprising (such as consisting of) the amino acid sequence of any one ofSEQ ID NOs: 50-52. In some embodiments, the abTCR further comprises afirst signal peptide amino-terminal to the first antigen-binding domainand/or a second signal peptide amino-terminal to the secondantigen-binding domain, wherein the first and/or second signal peptidescomprise the amino acid sequence of SEQ ID NO: 49. In some embodiments,the TCRM is capable of recruiting at least one TCR-associated signalingmodule selected from the group consisting of CD3δε, CD3γε, and ζζ. Insome embodiments, the TCRM promotes abTCR-CD3 complex formation. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between residuesin the C_(H)1 and C_(L) antibody domains in the Fab-like antigen-bindingmodule. In some embodiments, the target antigen is a cell surfaceantigen. In some embodiments, the cell surface antigen is selected fromthe group consisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a target antigen comprising a) a first polypeptide chaincomprising, in order from amino terminus to carboxy terminus, a firstantigen-binding domain and a first TCRD comprising the amino acidsequence of SEQ ID NO: 19; and b) a second polypeptide chain comprising,in order from amino terminus to carboxy terminus, a secondantigen-binding domain and a second TCRD comprising the amino acidsequence of SEQ ID NO: 20; wherein the first antigen-binding domain andthe second antigen-binding domain form a Fab-like antigen-binding modulethat specifically binds the target antigen, wherein the first TCRD andthe second TCRD form a TCRM that is capable of recruiting at least oneTCR-associated signaling module. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the abTCR further comprises atleast one accessory intracellular domain comprising a) at least one Tcell costimulatory signaling sequence comprising (such as consisting of)the amino acid sequence of SEQ ID NO: 70 or 71; and/or b) an epitope tagcomprising (such as consisting of) the amino acid sequence of any one ofSEQ ID NOs: 50-52. In some embodiments, the abTCR further comprises afirst signal peptide amino-terminal to the first antigen-binding domainand/or a second signal peptide amino-terminal to the secondantigen-binding domain, wherein the first and/or second signal peptidescomprise the amino acid sequence of SEQ ID NO: 49. In some embodiments,the TCRM is capable of recruiting at least one TCR-associated signalingmodule selected from the group consisting of CD3δε, CD3γε, and ζζ. Insome embodiments, the TCRM promotes abTCR-CD3 complex formation. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between residuesin the C_(H)1 and C_(L) antibody domains in the Fab-like antigen-bindingmodule. In some embodiments, the target antigen is a cell surfaceantigen. In some embodiments, the cell surface antigen is selected fromthe group consisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a target antigen comprising a) a first polypeptide chaincomprising, in order from amino terminus to carboxy terminus, a firstantigen-binding domain and a first TCRD comprising the amino acidsequence of SEQ ID NO: 21; and b) a second polypeptide chain comprising,in order from amino terminus to carboxy terminus, a secondantigen-binding domain and a second TCRD comprising the amino acidsequence of SEQ ID NO: 22; wherein the first antigen-binding domain andthe second antigen-binding domain form a Fab-like antigen-binding modulethat specifically binds the target antigen, wherein the first TCRD andthe second TCRD form a TCRM that is capable of recruiting at least oneTCR-associated signaling module. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the abTCR further comprises atleast one accessory intracellular domain comprising a) at least one Tcell costimulatory signaling sequence comprising (such as consisting of)the amino acid sequence of SEQ ID NO: 70 or 71; and/or b) an epitope tagcomprising (such as consisting of) the amino acid sequence of any one ofSEQ ID NOs: 50-52. In some embodiments, the abTCR further comprises afirst signal peptide amino-terminal to the first antigen-binding domainand/or a second signal peptide amino-terminal to the secondantigen-binding domain, wherein the first and/or second signal peptidescomprise the amino acid sequence of SEQ ID NO: 49. In some embodiments,the TCRM is capable of recruiting at least one TCR-associated signalingmodule selected from the group consisting of CD3δε, CD3γε, and ζζ. Insome embodiments, the TCRM promotes abTCR-CD3 complex formation. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between residuesin the C_(H)1 and C_(L) antibody domains in the Fab-like antigen-bindingmodule. In some embodiments, the target antigen is a cell surfaceantigen. In some embodiments, the cell surface antigen is selected fromthe group consisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01.

In some embodiments, there is provided an abTCR that specificallyrecognizes a complex comprising an AFP peptide and an MHC I proteincomprising a) a first polypeptide chain comprising a first abTCR domaincomprising the amino acid sequence of SEQ ID NO: 23; and b) a secondpolypeptide chain comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 24. In some embodiments, the firstpolypeptide chain and the second polypeptide chain are linked via one ormore disulfide bonds. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49.

In some embodiments, there is provided an abTCR that specificallyrecognizes a complex comprising an AFP peptide and an MHC I proteincomprising a) a first polypeptide chain comprising a first abTCR domaincomprising the amino acid sequence of SEQ ID NO: 25; and b) a secondpolypeptide chain comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 26. In some embodiments, the firstpolypeptide chain and the second polypeptide chain are linked via one ormore disulfide bonds. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49.

In some embodiments, there is provided an abTCR that specificallyrecognizes a complex comprising an AFP peptide and an MHC I proteincomprising a) a first polypeptide chain comprising a first abTCR domaincomprising the amino acid sequence of SEQ ID NO: 27; and b) a secondpolypeptide chain comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 28. In some embodiments, the firstpolypeptide chain and the second polypeptide chain are linked via one ormore disulfide bonds. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49.

In some embodiments, there is provided an abTCR that specificallyrecognizes a complex comprising an AFP peptide and an MHC I proteincomprising a) a first polypeptide chain comprising a first abTCR domaincomprising the amino acid sequence of SEQ ID NO: 29; and b) a secondpolypeptide chain comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 30. In some embodiments, the firstpolypeptide chain and the second polypeptide chain are linked via one ormore disulfide bonds. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49.

In some embodiments, there is provided an abTCR that specificallyrecognizes a complex comprising an AFP peptide and an MHC I proteincomprising a) a first polypeptide chain comprising a first abTCR domaincomprising the amino acid sequence of SEQ ID NO: 31; and b) a secondpolypeptide chain comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 32. In some embodiments, the firstpolypeptide chain and the second polypeptide chain are linked via one ormore disulfide bonds. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49.

In some embodiments, there is provided an abTCR that specificallyrecognizes a complex comprising an AFP peptide and an MHC I proteincomprising a) a first polypeptide chain comprising a first abTCR domaincomprising the amino acid sequence of SEQ ID NO: 33; and b) a secondpolypeptide chain comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 34. In some embodiments, the firstpolypeptide chain and the second polypeptide chain are linked via one ormore disulfide bonds. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49.

In some embodiments, there is provided an abTCR that specificallyrecognizes a complex comprising an AFP peptide and an MHC I proteincomprising a) a first polypeptide chain comprising a first abTCR domaincomprising the amino acid sequence of SEQ ID NO: 35; and b) a secondpolypeptide chain comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 36. In some embodiments, the firstpolypeptide chain and the second polypeptide chain are linked via one ormore disulfide bonds. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49.

In some embodiments, there is provided an abTCR that specificallyrecognizes a complex comprising an AFP peptide and an MHC I protein,comprising an antigen-binding module comprising a V_(H) antibody domaincomprising (and in some embodiments consisting of) the amino acidsequence of SEQ ID NO: 38, or a variant thereof having at least about95% (for example at least about any of 96%, 97%, 98%, or 99%) sequenceidentity, and a V_(L) antibody domain comprising (and in someembodiments consisting of) the amino acid sequence of SEQ ID NO: 40, ora variant thereof having at least about 95% (for example at least aboutany of 96%, 97%, 98%, or 99%) sequence identity.

Thus, in some embodiments, there is provided an abTCR that specificallyrecognizes a complex comprising an AFP peptide and an MHC I proteinaccording to any of the abTCRs described above, wherein the V_(H)antibody domain of the Fab-like antigen-binding module is replaced witha sequence comprising (and in some embodiments consisting of) the aminoacid sequence of SEQ ID NO: 38, or a variant thereof having at leastabout 95% (for example at least about any of 96%, 97%, 98%, or 99%)sequence identity, and wherein the V_(L) antibody domain of the Fab-likeantigen-binding module is replaced with a sequence comprising (and insome embodiments consisting of) the amino acid sequence of SEQ ID NO:40, or a variant thereof having at least about 95% (for example at leastabout any of 96%, 97%, 98%, or 99%) sequence identity.

In some embodiments, there is provided an abTCR that specificallyrecognizes CD19 comprising a) a first polypeptide chain comprising afirst abTCR domain comprising the amino acid sequence of SEQ ID NO: 42;and b) a second polypeptide chain comprising a second abTCR domaincomprising the amino acid sequence of SEQ ID NO: 43. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via one or more disulfide bonds. In some embodiments,the abTCR further comprises at least one accessory intracellular domaincomprising a T cell costimulatory signaling sequence (such as from CD27,CD28, 4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such asHA, FLAG, or myc). In some embodiments, the epitope tag comprises anyone of the amino acid sequences of SEQ ID NOs: 50-52. In someembodiments, the first polypeptide chain further comprises a firstsignal peptide amino terminal to the first abTCR domain and/or thesecond polypeptide chain further comprises a second signal peptide aminoterminal to the second abTCR domain. In some embodiments, the firstand/or second signal peptides comprise (such as consist of) the aminoacid sequence of SEQ ID NO: 49.

In some embodiments, there is provided an abTCR that specificallyrecognizes CD19 comprising a) a first polypeptide chain comprising afirst abTCR domain comprising the amino acid sequence of SEQ ID NO: 42;and b) a second polypeptide chain comprising a second abTCR domaincomprising the amino acid sequence of SEQ ID NO: 54. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via one or more disulfide bonds. In some embodiments,the abTCR further comprises at least one accessory intracellular domaincomprising a T cell costimulatory signaling sequence (such as from CD27,CD28, 4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such asHA, FLAG, or myc). In some embodiments, the epitope tag comprises anyone of the amino acid sequences of SEQ ID NOs: 50-52. In someembodiments, the first polypeptide chain further comprises a firstsignal peptide amino terminal to the first abTCR domain and/or thesecond polypeptide chain further comprises a second signal peptide aminoterminal to the second abTCR domain. In some embodiments, the firstand/or second signal peptides comprise (such as consist of) the aminoacid sequence of SEQ ID NO: 49.

In some embodiments, there is provided an abTCR that specificallyrecognizes CD19 comprising a) a first polypeptide chain comprising afirst abTCR domain comprising the amino acid sequence of SEQ ID NO: 55;and b) a second polypeptide chain comprising a second abTCR domaincomprising the amino acid sequence of SEQ ID NO: 54. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via one or more disulfide bonds. In some embodiments,the abTCR further comprises at least one accessory intracellular domaincomprising a T cell costimulatory signaling sequence (such as from CD27,CD28, 4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such asHA, FLAG, or myc). In some embodiments, the epitope tag comprises anyone of the amino acid sequences of SEQ ID NOs: 50-52. In someembodiments, the first polypeptide chain further comprises a firstsignal peptide amino terminal to the first abTCR domain and/or thesecond polypeptide chain further comprises a second signal peptide aminoterminal to the second abTCR domain. In some embodiments, the firstand/or second signal peptides comprise (such as consist of) the aminoacid sequence of SEQ ID NO: 49.

In some embodiments, there is provided an abTCR that specificallyrecognizes CD19 comprising a) a first polypeptide chain comprising afirst abTCR domain comprising the amino acid sequence of SEQ ID NO: 56;and b) a second polypeptide chain comprising a second abTCR domaincomprising the amino acid sequence of SEQ ID NO: 54. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via one or more disulfide bonds. In some embodiments,the abTCR further comprises at least one accessory intracellular domaincomprising a T cell costimulatory signaling sequence (such as from CD27,CD28, 4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such asHA, FLAG, or myc). In some embodiments, the epitope tag comprises anyone of the amino acid sequences of SEQ ID NOs: 50-52. In someembodiments, the first polypeptide chain further comprises a firstsignal peptide amino terminal to the first abTCR domain and/or thesecond polypeptide chain further comprises a second signal peptide aminoterminal to the second abTCR domain. In some embodiments, the firstand/or second signal peptides comprise (such as consist of) the aminoacid sequence of SEQ ID NO: 49.

In some embodiments, there is provided an abTCR according to any of theembodiments described herein that specifically recognizes CD19comprising an antigen-binding module comprising a V_(H) antibody domaincomprising (and in some embodiments consisting of) the amino acidsequence of SEQ ID NO: 45, or a variant thereof having at least about95% (for example at least about any of 96%, 97%, 98%, or 99%) sequenceidentity, and a V_(L) antibody domain comprising (and in someembodiments consisting of) the amino acid sequence of SEQ ID NO: 46, ora variant thereof having at least about 95% (for example at least aboutany of 96%, 97%, 98%, or 99%) sequence identity.

In some embodiments, there is provided an abTCR according to any of theembodiments described herein that specifically recognizes CD19comprising an antigen-binding module comprising a V_(H) antibody domaincomprising (and in some embodiments consisting of) the amino acidsequence of SEQ ID NO: 45, or a variant thereof having at least about95% (for example at least about any of 96%, 97%, 98%, or 99%) sequenceidentity, and a V_(L) antibody domain comprising (and in someembodiments consisting of) the amino acid sequence of SEQ ID NO: 57, ora variant thereof having at least about 95% (for example at least aboutany of 96%, 97%, 98%, or 99%) sequence identity.

In some embodiments, there is provided an abTCR according to any of theembodiments described herein that specifically recognizes CD19comprising an antigen-binding module comprising a V_(H) antibody domaincomprising (and in some embodiments consisting of) the amino acidsequence of SEQ ID NO: 58, or a variant thereof having at least about95% (for example at least about any of 96%, 97%, 98%, or 99%) sequenceidentity, and a V_(L) antibody domain comprising (and in someembodiments consisting of) the amino acid sequence of SEQ ID NO: 57, ora variant thereof having at least about 95% (for example at least aboutany of 96%, 97%, 98%, or 99%) sequence identity.

In some embodiments, there is provided an abTCR according to any of theembodiments described herein that specifically recognizes CD19comprising an antigen-binding module comprising a V_(H) antibody domaincomprising (and in some embodiments consisting of) the amino acidsequence of SEQ ID NO: 59, or a variant thereof having at least about95% (for example at least about any of 96%, 97%, 98%, or 99%) sequenceidentity, and a V_(L) antibody domain comprising (and in someembodiments consisting of) the amino acid sequence of SEQ ID NO: 57, ora variant thereof having at least about 95% (for example at least aboutany of 96%, 97%, 98%, or 99%) sequence identity.

In some embodiments, there is provided an abTCR according to any of theembodiments described herein that specifically recognizes a complexcomprising an NY-ESO-1 157-165 peptide and an MHC I protein comprisingan antigen-binding module comprising a V_(H) antibody domain comprising(and in some embodiments consisting of) the amino acid sequence of SEQID NO: 72, or a variant thereof having at least about 95% (for exampleat least about any of 96%, 97%, 98%, or 99%) sequence identity, and aV_(L) antibody domain comprising (and in some embodiments consisting of)the amino acid sequence of SEQ ID NO: 73, or a variant thereof having atleast about 95% (for example at least about any of 96%, 97%, 98%, or99%) sequence identity.

In some embodiments, there is provided an abTCR comprising a firstantigen-binding module that competes for binding to a target antigenwith a second antigen-binding module according to any of the abTCRsdescribed herein. In some embodiments, the first antigen-binding modulebinds to the same, or substantially the same, epitope as the secondantigen-binding module. In some embodiments, binding of the firstantigen-binding module to the target antigen inhibits binding of thesecond antigen-binding module to the target antigen by at least about70% (such as by at least about any of 75%, 80%, 85%, 90%, 95%, 98% or99%), or vice versa. In some embodiments, the first antigen-bindingmodule and the second antigen-binding module cross-compete for bindingto the target antigen, i.e., each of the first and secondantigen-binding modules competes with the other for binding to thetarget antigen.

In some embodiments, there is provided an abTCR according to any of theabTCRs described herein, wherein the V_(L) and V_(H) domains areinterchanged, such that the first antigen-binding domain comprises V_(L)and C_(H)1 antibody domains and the second antigen-binding domaincomprises V_(H) and C_(L) antibody domains.

In some embodiments, there is provided a complex comprising an abTCRaccording to any of the abTCRs described herein and at least onesignaling module selected from the group consisting of CD3δε, CD3γε, andζζ. In some embodiments, the complex comprises each of CD3δε, CD3γε, andThus, in some embodiments, there is provided a complex comprising theabTCR, CD3δε, CD3γε, and

The different aspects are discussed in various sections below in furtherdetail.

Nucleic Acids

Nucleic acid molecules encoding the abTCRs are also contemplated. Insome embodiments, according to any of the abTCRs described herein, thereis provided a nucleic acid (or a set of nucleic acids) encoding theabTCR.

The present invention also provides vectors in which a nucleic acid ofthe present invention is inserted.

In brief summary, the expression of an abTCR by a nucleic acid encodingthe abTCR can be achieved by inserting the nucleic acid into anappropriate expression vector, such that the nucleic acid is operablylinked to 5′ and 3′ regulatory elements, including for example apromoter (e.g., a lymphocyte-specific promoter) and a 3′ untranslatedregion (UTR). The vectors can be suitable for replication andintegration in eukaryotic host cells. Typical cloning and expressionvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The nucleic acids of the present invention may also be used for nucleicacid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In some embodiments, the inventionprovides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to, a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers (see, e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In someembodiments, lentivirus vectors are used. Vectors derived fromretroviruses such as the lentivirus are suitable tools to achievelong-term gene transfer since they allow long-term, stable integrationof a transgene and its propagation in daughter cells. Lentiviral vectorshave the added advantage over vectors derived from onco-retrovirusessuch as murine leukemia viruses in that they can transducenon-proliferating cells, such as hepatocytes. They also have the addedadvantage of low immunogenicity.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter.

Further, the invention should not be limited to the use of constitutivepromoters. Inducible promoters are also contemplated as part of theinvention. The use of an inducible promoter provides a molecular switchcapable of turning on expression of the polynucleotide sequence which itis operatively linked when such expression is desired, or turning offthe expression when expression is not desired. Exemplary induciblepromoter systems for use in eukaryotic cells include, but are notlimited to, hormone-regulated elements (e.g., see Mader, S. and White,J. H. (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), syntheticligand-regulated elements (see, e.g., Spencer, D. M. et al 1993) Science262: 1019-1024) and ionizing radiation-regulated elements (e.g., seeManome, Y. et al. (1993) Biochemistry 32: 10607-10613; Datta, R. et al.(1992) Proc. Natl. Acad. Sci. USA 89: 1014-10153). Further exemplaryinducible promoter systems for use in in vitro or in vivo mammaliansystems are reviewed in Gingrich et al. (1998) Annual Rev. Neurosci21:377-405.

An exemplary inducible promoter system for use in the present inventionis the Tet system. Such systems are based on the Tet system described byGossen et al. (1993). In an exemplary embodiment, a polynucleotide ofinterest is under the control of a promoter that comprises one or moreTet operator (TetO) sites. In the inactive state, Tet repressor (TetR)will bind to the TetO sites and repress transcription from the promoter.In the active state, e.g., in the presence of an inducing agent such astetracycline (Tc), anhydrotetracycline, doxycycline (Dox), or an activeanalog thereof, the inducing agent causes release of TetR from TetO,thereby allowing transcription to take place. Doxycycline is a member ofthe tetracycline family of antibiotics having the chemical name of1-dimethylamino-2,4a,5,7,12-pentahydroxy-11-methyl-4,6-dioxo-1,4a,11,11a,12,12a-hexahydrotetracene-3-carboxamide.

In one embodiment, a TetR is codon-optimized for expression in mammaliancells, e.g., murine or human cells. Most amino acids are encoded by morethan one codon due to the degeneracy of the genetic code, allowing forsubstantial variations in the nucleotide sequence of a given nucleicacid without any alteration in the amino acid sequence encoded by thenucleic acid. However, many organisms display differences in codonusage, also known as “codon bias” (i.e., bias for use of a particularcodon(s) for a given amino acid). Codon bias often correlates with thepresence of a predominant species of tRNA for a particular codon, whichin turn increases efficiency of mRNA translation. Accordingly, a codingsequence derived from a particular organism (e.g., a prokaryote) may betailored for improved expression in a different organism (e.g., aeukaryote) through codon optimization.

Other specific variations of the Tet system include the following“Tet-Off” and “Tet-On” systems. In the Tet-Off system, transcription isinactive in the presence of Tc or Dox. In that system, atetracycline-controlled transactivator protein (tTA), which is composedof TetR fused to the strong transactivating domain of VP16 from Herpessimplex virus, regulates expression of a target nucleic acid that isunder transcriptional control of a tetracycline-responsive promoterelement (TRE). The TRE is made up of TetO sequence concatamers fused toa promoter (commonly the minimal promoter sequence derived from thehuman cytomegalovirus (hCMV) immediate-early promoter). In the absenceof Tc or Dox, tTA binds to the TRE and activates transcription of thetarget gene. In the presence of Tc or Dox, tTA cannot bind to the TRE,and expression from the target gene remains inactive.

Conversely, in the Tet-On system, transcription is active in thepresence of Tc or Dox. The Tet-On system is based on a reversetetracycline-controlled transactivator, rtTA. Like tTA, rtTA is a fusionprotein comprised of the TetR repressor and the VP16 transactivationdomain. However, a four amino acid change in the TetR DNA binding moietyalters rtTA's binding characteristics such that it can only recognizethe tetO sequences in the TRE of the target transgene in the presence ofDox. Thus, in the Tet-On system, transcription of the TRE-regulatedtarget gene is stimulated by rtTA only in the presence of Dox.

Another inducible promoter system is the lac repressor system from E.coli. (See, Brown et al., Cell 49:603-612 (1987). The lac repressorsystem functions by regulating transcription of a polynucleotide ofinterest operably linked to a promoter comprising the lac operator(lacO). The lac repressor (lacR) binds to LacO, thus preventingtranscription of the polynucleotide of interest. Expression of thepolynucleotide of interest is induced by a suitable inducing agent,e.g., isopropyl-β-D-thiogalactopyranoside (IPTG).

In order to assess the expression of a polypeptide or portions thereof,the expression vector to be introduced into a cell can also containeither a selectable marker gene or a reporter gene or both to facilitateidentification and selection of expressing cells from the population ofcells sought to be transfected or infected through viral vectors. Inother aspects, the selectable marker may be carried on a separate pieceof DNA and used in a co-transfection procedure. Both selectable markersand reporter genes may be flanked with appropriate regulatory sequencesto enable expression in the host cells. Useful selectable markersinclude, for example, antibiotic-resistance genes, such as neo and thelike.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,β-galactosidase, chloramphenicol acetyl transferase, secreted alkalinephosphatase, or the green fluorescent protein gene (e.g., Ui-Tel et al.,2000 FEBS Letters 479: 79-82). Suitable expression systems are wellknown and may be prepared using known techniques or obtainedcommercially. In general, the construct with the minimal 5′ flankingregion showing the highest level of expression of reporter gene isidentified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

In some embodiments, there is provided nucleic acid encoding an abTCRaccording to any of the abTCRs described herein. In some embodiments,the nucleic acid encoding the abTCR comprises a first nucleic acidsequence encoding the first polypeptide chain of the abTCR and a secondnucleic acid sequence encoding the second polypeptide chain of theabTCR. In some embodiments, the first nucleic acid sequence is locatedon a first vector and the second nucleic acid sequence is located on asecond vector. In some embodiments, the first and second nucleic acidsequences are located on the same vector. Vectors may be selected, forexample, from the group consisting of mammalian expression vectors andviral vectors (such as those derived from retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses). In someembodiments, the first nucleic acid sequence is under the control of afirst promoter and the second nucleic acid sequence is under the controlof a second promoter. In some embodiments, the first and secondpromoters have the same sequence. In some embodiments, the first andsecond promoters have different sequences. In some embodiments, thefirst and second nucleic acid sequences are expressed as a singletranscript under the control of a single promoter in a multicistronic(such as a bicistronic) vector. See for example Kim, J H, et al., PLoSOne 6(4):e18556, 2011. In some embodiments, the first, second, and/orsingle promoters are inducible. In some embodiments, the first nucleicacid sequence has an expression level in a host cell (such as a T cell)that is about the same as the expression level of the second nucleicacid sequence in the host cell. In some embodiments, the first nucleicacid sequence has an expression level in a host cell (such as a T cell)that is at least about two (such as at least about any of 2, 3, 4, 5, ormore) times the expression level of the second nucleic acid sequence inthe host cell. In some embodiments, the first nucleic acid sequence hasan expression level in a host cell (such as a T cell) that is no morethan about ½ (such as no more than about any of ½, ⅓, ¼, ⅕ or less)times the expression level of the second nucleic acid sequence in thehost cell. Expression can be determined at the mRNA or protein level.The level of mRNA expression can be determined by measuring the amountof mRNA transcribed from the nucleic acid using various well-knownmethods, including Northern blotting, quantitative RT-PCR, microarrayanalysis and the like. The level of protein expression can be measuredby known methods including immunocytochemical staining, enzyme-linkedimmunosorbent assay (ELISA), western blot analysis, luminescent assays,mass spectrometry, high performance liquid chromatography, high-pressureliquid chromatography-tandem mass spectrometry, and the like.

Thus, in some embodiments, there is provided nucleic acid encoding anabTCR according to any of the abTCRs described herein comprising a) afirst nucleic acid sequence encoding the first polypeptide chain of theabTCR, and b) a second nucleic acid sequence encoding the secondpolypeptide chain of the abTCR, wherein the first nucleic acid sequenceis located on a first vector (such as a lentiviral vector) and operablylinked to a first promoter and the second nucleic acid sequence islocated on a second vector (such as a lentiviral vector) and operablylinked to a second promoter. In some embodiments, the first and secondpromoters have the same sequence. In some embodiments, the first andsecond promoters have different sequences. In some embodiments, thefirst and/or second promoters are inducible. In some embodiments, thefirst nucleic acid sequence has an expression level in a host cell (suchas a T cell) that is about the same as the expression level of thesecond nucleic acid sequence in the host cell. In some embodiments, thefirst nucleic acid sequence has an expression level in a host cell (suchas a T cell) that is at least about two (such as at least about any of2, 3, 4, 5, or more) times the expression level of the second nucleicacid sequence in the host cell. In some embodiments, the first nucleicacid sequence has an expression level in a host cell (such as a T cell)that is no more than about ½ (such as no more than about any of ½, ⅓, ¼,⅕ or less) times the expression level of the second nucleic acidsequence in the host cell. In some embodiments, the first and/or secondvectors are viral vectors (such as lentiviral vectors).

In some embodiments, there is provided a vector (such as a lentiviralvector) comprising nucleic acid encoding an abTCR according to any ofthe abTCRs described herein comprising a) a first promoter operablylinked to a first nucleic acid sequence encoding the first polypeptidechain of the abTCR; and b) a second promoter operably linked to a secondnucleic acid sequence encoding the second polypeptide chain of theabTCR. In some embodiments, the first and second promoters have the samesequence. In some embodiments, the first and second promoters havedifferent sequences. In some embodiments, the first and/or secondpromoters are inducible. In some embodiments, the first nucleic acidsequence has an expression level in a host cell (such as a T cell) thatis about the same as the expression level of the second nucleic acidsequence in the host cell. In some embodiments, the first nucleic acidsequence has an expression level in a host cell (such as a T cell) thatis at least about two (such as at least about any of 2, 3, 4, 5, ormore) times the expression level of the second nucleic acid sequence inthe host cell. In some embodiments, the first nucleic acid sequence hasan expression level in a host cell (such as a T cell) that is no morethan about ½ (such as no more than about any of ½, ⅓, ¼, ⅕ or less)times the expression level of the second nucleic acid sequence in thehost cell. In some embodiments, the vector is a viral vector (such as alentiviral vector).

In some embodiments, there is provided a vector (such as a lentiviralvector) comprising nucleic acid encoding an abTCR according to any ofthe abTCRs described herein comprising a) a first nucleic acid sequenceencoding the first polypeptide chain of the abTCR; and b) a secondnucleic acid sequence encoding the second polypeptide chain of theabTCR; wherein the first and second nucleic acid sequences are under thecontrol of a single promoter. In some embodiments, the promoter isoperably linked to the 5′ end of the first nucleic acid sequence, andthere is nucleic acid linker selected from the group consisting of aninternal ribosomal entry site (IRES) and a nucleic acid encoding aself-cleaving 2A peptide (such as P2A, T2A, E2A, or F2A) linking the 3′end of first nucleic acid sequence to the 5′ end of the second nucleicacid sequence, wherein the first nucleic acid sequence and the secondnucleic acid sequence are transcribed as a single RNA under the controlof the promoter. In some embodiments, the promoter is operably linked tothe 5′ end of the second nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of second nucleicacid sequence to the 5′ end of the first nucleic acid sequence, whereinthe first nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, the promoter is inducible. In some embodiments, the vectoris a viral vector (such as a lentiviral vector).

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). In some embodiments, the introduction of a polynucleotideinto a host cell is carried out by calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human, cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virus1, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

abTCR Effector Cells

In some embodiments, there is provided an effector cell (such as a Tcell) presenting on its surface an abTCR according to any of the abTCRsdescribed herein. In some embodiments, the effector cell comprises anucleic acid encoding the abTCR, wherein the abTCR is expressed from thenucleic acid and localized to the effector cell surface. In someembodiments, the abTCR is exogenously expressed and combined with theeffector cell. In some embodiments, the effector cell is a T cell. Insome embodiments, the effector cell is selected from the groupconsisting of a cytotoxic T cell, a helper T cell, a natural killer Tcell, and a suppressor T cell. In some embodiments, the effector celldoes not express the TCR subunits from which the TCRDs of the abTCR arederived. For example, in some embodiments, the effector cell is an αβ Tcell and the TCRDs of the introduced abTCR comprise sequences derivedfrom TCR δ and γ chains, or the T cell is a γδ T cell and the TCRDs ofthe introduced abTCR comprise sequences derived from TCR α and β chains.In some embodiments, the effector cell is modified to block or decreasethe expression of one or both of the endogenous TCR subunits from whichthe TCRDs of the abTCR are derived. For example, in some embodiments,the effector cell is an αβ T cell modified to block or decrease theexpression of the TCR α and/or β chains and the TCRDs of the introducedabTCR comprise sequences derived from TCR α and β chains, or theeffector cell is a γδ T cell modified to block or decrease theexpression of the TCR γ and/or δ chains and the TCRDs of the introducedabTCR comprise sequences derived from TCR γ and δ chains. Modificationsof cells to disrupt gene expression include any such techniques known inthe art, including for example RNA interference (e.g., siRNA, shRNA,miRNA), gene editing (e.g., CRISPR- or TALEN-based gene knockout), andthe like. For example, in some embodiments, there is provided aneffector cell (such as a T cell) comprising a nucleic acid encoding anabTCR according to any of the abTCRs described herein, wherein the abTCRis expressed from the nucleic acid and localized to the effector cellsurface. In some embodiments, the nucleic acid encoding the abTCRcomprises a first nucleic acid sequence encoding the first polypeptidechain of the abTCR and a second nucleic acid sequence encoding thesecond polypeptide chain of the abTCR. In some embodiments, the firstnucleic acid sequence is located on a first vector and the secondnucleic acid sequence is located on a second vector. In someembodiments, the first and second nucleic acid sequences are located onthe same vector. Vectors may be selected, for example, from the groupconsisting of mammalian expression vectors and viral vectors (such asthose derived from retroviruses, adenoviruses, adeno-associated viruses,herpes viruses, and lentiviruses). In some embodiments, one or more ofthe vectors is integrated into the host genome of the effector cell. Insome embodiments, the first nucleic acid sequence is under the controlof a first promoter and the second nucleic acid sequence is under thecontrol of a second promoter. In some embodiments, the first and secondpromoters have the same sequence. In some embodiments, the first andsecond promoters have different sequences. In some embodiments, thefirst and second nucleic acids are under the control of a singlepromoter. In some embodiments, the first, second, and/or singlepromoters are inducible. In some embodiments, the expression of thefirst polypeptide chain is about the same as the expression of thesecond polypeptide chain. In some embodiments, the expression of thefirst polypeptide chain is at least about two (such as at least aboutany of 2, 3, 4, 5, or more) times the expression of the secondpolypeptide chain. In some embodiments, the expression of the firstpolypeptide chain is no more than about ½ (such as no more than aboutany of ½, ⅓, ¼, ⅕ or less) times the expression of the secondpolypeptide chain. Expression can be determined at the mRNA or proteinlevel. The level of mRNA expression can be determined by measuring theamount of mRNA transcribed from the nucleic acid using variouswell-known methods, including Northern blotting, quantitative RT-PCR,microarray analysis and the like. The level of protein expression can bemeasured by known methods including immunocytochemical staining,enzyme-linked immunosorbent assay (ELISA), western blot analysis,luminescent assays, mass spectrometry, high performance liquidchromatography, high-pressure liquid chromatography-tandem massspectrometry, and the like. In some embodiments, the effector cell isselected from the group consisting of a cytotoxic T cell, a helper Tcell, a natural killer T cell, and a suppressor T cell.

Thus, in some embodiments, there is provided an abTCR effector cell(such as a T cell) expressing on its surface an abTCR according to anyof the abTCRs described herein, wherein the abTCR effector cellcomprises a) a first nucleic acid comprising a first promoter operablylinked to a nucleic acid sequence encoding the first polypeptide chainof the abTCR and b) a second nucleic acid comprising a second promoteroperably linked to a nucleic acid sequence encoding the secondpolypeptide chain of the abTCR, wherein the first polypeptide chain isexpressed from the first nucleic acid and the second polypeptide chainis expressed from the second nucleic acid to form the abTCR, and whereinthe abTCR localizes to the surface of the effector cell. In someembodiments, the first and second promoters have the same sequence. Insome embodiments, the first and second promoters have differentsequences. In some embodiments, the first and/or second promoters areinducible. In some embodiments, the expression of the first polypeptidechain is about the same as the expression of the second polypeptidechain. In some embodiments, the expression of the first polypeptidechain is at least about two (such as at least about any of 2, 3, 4, 5,or more) times the expression of the second polypeptide chain. In someembodiments, the expression of the first polypeptide chain is no morethan about ½ (such as no more than about any of ½, ⅓, ¼, ⅕ or less)times the expression of the second polypeptide chain. In someembodiments, the effector cell does not express the TCR subunits fromwhich the TCRDs of the abTCR are derived. For example, in someembodiments, the effector cell is an αβ T cell and the TCRDs of theintroduced abTCR comprise sequences derived from TCR δ and γ chains, orthe effector cell is a γδ T cell and the TCRDs of the introduced abTCRcomprise sequences derived from TCR α and β chains. In some embodiments,the effector cell is modified to block or decrease the expression of oneor both of the endogenous TCR subunits from which the TCRDs of the abTCRare derived. For example, in some embodiments, the effector cell is anαβ T cell modified to block or decrease the expression of the TCR αand/or β chains and the TCRDs of the introduced abTCR comprise sequencesderived from TCR α and β chains, or the effector cell is a γδ T cellmodified to block or decrease the expression of the TCR γ and/or δchains and the TCRDs of the introduced abTCR comprise sequences derivedfrom TCR γ and δ chains. In some embodiments, the effector cell isselected from the group consisting of a cytotoxic T cell, a helper Tcell, a natural killer T cell, and a suppressor T cell. In someembodiments, the vector is a viral vector (such as a lentiviral vector)integrated into the host genome of the effector cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR according to any of theabTCRs described herein, wherein the abTCR effector cell comprises a) afirst vector comprising a first promoter operably linked to a firstnucleic acid sequence encoding the first polypeptide chain of the abTCRand b) a second vector comprising a second promoter operably linked to asecond nucleic acid sequence encoding the second polypeptide chain ofthe abTCR, wherein the first polypeptide chain is expressed from thefirst nucleic acid sequence and the second polypeptide chain isexpressed from the second nucleic acid sequence to form the abTCR, andwherein the abTCR localizes to the surface of the effector cell. In someembodiments, the first and second promoters have the same sequence. Insome embodiments, the first and second promoters have differentsequences. In some embodiments, the first and/or second promoters areinducible. In some embodiments, the expression of the first polypeptidechain is about the same as the expression of the second polypeptidechain. In some embodiments, the expression of the first polypeptidechain is at least about two (such as at least about any of 2, 3, 4, 5,or more) times the expression of the second polypeptide chain. In someembodiments, the expression of the first polypeptide chain is no morethan about ½ (such as no more than about any of ½, ⅓, ¼, ⅕ or less)times the expression of the second polypeptide chain. In someembodiments, the effector cell does not express the TCR subunits fromwhich the TCRDs of the abTCR are derived. For example, in someembodiments, the effector cell is an αβ T cell and the TCRDs of theintroduced abTCR comprise sequences derived from TCR δ and γ chains, orthe effector cell is a γδ T cell and the TCRDs of the introduced abTCRcomprise sequences derived from TCR α and β chains. In some embodiments,the effector cell is modified to block or decrease the expression of oneor both of the endogenous TCR subunits from which the TCRDs of the abTCRare derived. For example, in some embodiments, the effector cell is anαβ T cell modified to block or decrease the expression of the TCR αand/or β chains and the TCRDs of the introduced abTCR comprise sequencesderived from TCR α and β chains, or the effector cell is a γδ T cellmodified to block or decrease the expression of the TCR γ and/or δchains and the TCRDs of the introduced abTCR comprise sequences derivedfrom TCR γ and δ chains. In some embodiments, the effector cell isselected from the group consisting of a cytotoxic T cell, a helper Tcell, a natural killer T cell, and a suppressor T cell. In someembodiments, the first and second vectors are viral vectors (such aslentiviral vectors) integrated into the host genome of the effectorcell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR according to any of theabTCRs described herein, wherein the abTCR effector cell comprises avector comprising a) a first promoter operably linked to a first nucleicacid sequence encoding the first polypeptide chain of the abTCR and b) asecond promoter operably linked to a second nucleic acid sequenceencoding the second polypeptide chain of the abTCR, wherein the firstpolypeptide chain is expressed from the first nucleic acid sequence andthe second polypeptide chain is expressed from the second nucleic acidsequence to form the abTCR, and wherein the abTCR localizes to thesurface of the effector cell. In some embodiments, the first and secondpromoters have the same sequence. In some embodiments, the first andsecond promoters have different sequences. In some embodiments, thefirst and/or second promoters are inducible. In some embodiments, theexpression of the first polypeptide chain is about the same as theexpression of the second polypeptide chain. In some embodiments, theexpression of the first polypeptide chain is at least about two (such asat least about any of 2, 3, 4, 5, or more) times the expression of thesecond polypeptide chain. In some embodiments, the expression of thefirst polypeptide chain is no more than about ½ (such as no more thanabout any of ½, ⅓, ¼, ⅕ or less) times the expression of the secondpolypeptide chain. In some embodiments, the effector cell does notexpress the TCR subunits from which the TCRDs of the abTCR are derived.For example, in some embodiments, the effector cell is an αβ T cell andthe TCRDs of the introduced abTCR comprise sequences derived from TCR δand γ chains, or the effector cell is a γδ T cell and the TCRDs of theintroduced abTCR comprise sequences derived from TCR α and β chains. Insome embodiments, the effector cell is modified to block or decrease theexpression of one or both of the endogenous TCR subunits from which theTCRDs of the abTCR are derived. For example, in some embodiments, theeffector cell is an αβ T cell modified to block or decrease theexpression of the TCR α and/or β chains and the TCRDs of the introducedabTCR comprise sequences derived from TCR α and β chains, or theeffector cell is a γδ T cell modified to block or decrease theexpression of the TCR γ and/or δ chains and the TCRDs of the introducedabTCR comprise sequences derived from TCR γ and δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell. In some embodiments, the first and second vectors areviral vectors (such as lentiviral vectors) integrated into the hostgenome of the effector cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR according to any of theabTCRs described herein, wherein the abTCR effector cell comprises ahost genome-integrated lentiviral vector comprising a) a first promoteroperably linked to a first nucleic acid sequence encoding the firstpolypeptide chain of the abTCR and b) a second promoter operably linkedto a second nucleic acid sequence encoding the second polypeptide chainof the abTCR, wherein the first polypeptide chain is expressed from thefirst nucleic acid sequence and the second polypeptide chain isexpressed from the second nucleic acid sequence to form the abTCR, andwherein the abTCR localizes to the surface of the effector cell. In someembodiments, the first and second promoters have the same sequence. Insome embodiments, the first and second promoters have differentsequences. In some embodiments, the first and/or second promoters areinducible. In some embodiments, the expression of the first polypeptidechain is about the same as the expression of the second polypeptidechain. In some embodiments, the expression of the first polypeptidechain is at least about two (such as at least about any of 2, 3, 4, 5,or more) times the expression of the second polypeptide chain. In someembodiments, the expression of the first polypeptide chain is no morethan about ½ (such as no more than about any of ½, ⅓, ¼, ⅕ or less)times the expression of the second polypeptide chain. In someembodiments, the effector cell does not express the TCR subunits fromwhich the TCRDs of the abTCR are derived. For example, in someembodiments, the effector cell is an αβ T cell and the TCRDs of theintroduced abTCR comprise sequences derived from TCR δ and γ chains, orthe effector cell is a γδ T cell and the TCRDs of the introduced abTCRcomprise sequences derived from TCR α and β chains. In some embodiments,the effector cell is modified to block or decrease the expression of oneor both of the endogenous TCR subunits from which the TCRDs of the abTCRare derived. For example, in some embodiments, the effector cell is anαβ T cell modified to block or decrease the expression of the TCR αand/or β chains and the TCRDs of the introduced abTCR comprise sequencesderived from TCR α and β chains, or the effector cell is a γδ T cellmodified to block or decrease the expression of the TCR γ and/or δchains and the TCRDs of the introduced abTCR comprise sequences derivedfrom TCR γ and δ chains. In some embodiments, the effector cell ismodified to block or decrease the expression of one or both of theendogenous TCR chains. In some embodiments, the effector cell isselected from the group consisting of a cytotoxic T cell, a helper Tcell, a natural killer T cell, and a suppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR according to any of theabTCRs described herein, wherein the abTCR effector cell comprises avector comprising a) a first nucleic acid sequence encoding the firstpolypeptide chain of the abTCR and b) a second nucleic acid sequenceencoding the second polypeptide chain of the abTCR, wherein the firstand second nucleic acid sequences are under the control of a singlepromoter, wherein the first polypeptide chain is expressed from thefirst nucleic acid sequence and the second polypeptide chain isexpressed from the second nucleic acid sequence to form the abTCR, andwherein the abTCR localizes to the surface of the effector cell. In someembodiments, the promoter is operably linked to the 5′ end of the firstnucleic acid sequence, and there is nucleic acid linker selected fromthe group consisting of an internal ribosomal entry site (IRES) and anucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A, E2A,or F2A) linking the 3′ end of first nucleic acid sequence to the 5′ endof the second nucleic acid sequence, wherein the first nucleic acidsequence and the second nucleic acid sequence are transcribed as asingle RNA under the control of the promoter. In some embodiments, thepromoter is operably linked to the 5′ end of the second nucleic acidsequence, and there is nucleic acid linker selected from the groupconsisting of an internal ribosomal entry site (IRES) and a nucleic acidencoding a self-cleaving 2A peptide (such as P2A, T2A, E2A, or F2A)linking the 3′ end of second nucleic acid sequence to the 5′ end of thefirst nucleic acid sequence, wherein the first nucleic acid sequence andthe second nucleic acid sequence are transcribed as a single RNA underthe control of the promoter. In some embodiments, the promoter isinducible. In some embodiments, the effector cell does not express theTCR subunits from which the TCRDs of the abTCR are derived. For example,in some embodiments, the effector cell is an αβ T cell and the TCRDs ofthe introduced abTCR comprise sequences derived from TCR δ and γ chains,or the effector cell is a γδ T cell and the TCRDs of the introducedabTCR comprise sequences derived from TCR α and β chains. In someembodiments, the effector cell is modified to block or decrease theexpression of one or both of the endogenous TCR subunits from which theTCRDs of the abTCR are derived. For example, in some embodiments, theeffector cell is an αβ T cell modified to block or decrease theexpression of the TCR α and/or β chains and the TCRDs of the introducedabTCR comprise sequences derived from TCR α and β chains, or theeffector cell is a γδ T cell modified to block or decrease theexpression of the TCR γ and/or δ chains and the TCRDs of the introducedabTCR comprise sequences derived from TCR γ and δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell. In some embodiments, the vector is a viral vector(such as a lentiviral vector) integrated into the host genome of theeffector cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR according to any of theabTCRs described herein, wherein the abTCR effector cell comprises ahost genome-integrated lentiviral vector comprising a) a first nucleicacid sequence encoding the first polypeptide chain of the abTCR and b) asecond nucleic acid sequence encoding the second polypeptide chain ofthe abTCR, wherein the first and second nucleic acid sequences are underthe control of a single promoter, wherein the first polypeptide chain isexpressed from the first nucleic acid sequence and the secondpolypeptide chain is expressed from the second nucleic acid sequence toform the abTCR, and wherein the abTCR localizes to the surface of theeffector cell. In some embodiments, there is a promoter operably linkedto the 5′ end of the first nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of first nucleic acidsequence to the 5′ end of the second nucleic acid sequence, wherein thefirst nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, there is a promoter operably linked to the 5′ end of thesecond nucleic acid sequence, and there is nucleic acid linker selectedfrom the group consisting of an internal ribosomal entry site (IRES) anda nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A,E2A, or F2A) linking the 3′ end of second nucleic acid sequence to the5′ end of the first nucleic acid sequence, wherein the first nucleicacid sequence and the second nucleic acid sequence are transcribed as asingle RNA under the control of the promoter. In some embodiments, thepromoter is inducible. In some embodiments, the effector cell does notexpress the TCR subunits from which the TCRDs of the abTCR are derived.For example, in some embodiments, the effector cell is an αβ T cell andthe TCRDs of the introduced abTCR comprise sequences derived from TCR δand γ chains, or the effector cell is a γδ T cell and the TCRDs of theintroduced abTCR comprise sequences derived from TCR α and β chains. Insome embodiments, the effector cell is modified to block or decrease theexpression of one or both of the endogenous TCR subunits from which theTCRDs of the abTCR are derived. For example, in some embodiments, theeffector cell is an αβ T cell modified to block or decrease theexpression of the TCR α and/or β chains and the TCRDs of the introducedabTCR comprise sequences derived from TCR α and β chains, or theeffector cell is a γδ T cell modified to block or decrease theexpression of the TCR γ and/or δ chains and the TCRDs of the introducedabTCR comprise sequences derived from TCR γ and δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain comprising, in orderfrom amino terminus to carboxy terminus, a first antigen-binding domainand a first TCRD comprising the amino acid sequence of SEQ ID NO: 15;and b) a second nucleic acid sequence encoding a second polypeptidechain comprising, in order from amino terminus to carboxy terminus, asecond antigen-binding domain and a second TCRD comprising the aminoacid sequence of SEQ ID NO: 16; wherein the first antigen-binding domainand the second antigen-binding domain form a Fab-like antigen-bindingmodule that specifically binds the target antigen, wherein the firstTCRD and the second TCRD form a TCRM that is capable of recruiting atleast one TCR-associated signaling module. In some embodiments, theFab-like antigen-binding module is human, humanized, chimeric,semi-synthetic, or fully synthetic. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga) at least one T cell costimulatory signaling sequence comprising (suchas consisting of) the amino acid sequence of SEQ ID NO: 70 or 71; and/orb) an epitope tag comprising (such as consisting of) the amino acidsequence of any one of SEQ ID NOs: 50-52. In some embodiments, the abTCRfurther comprises a first signal peptide amino-terminal to the firstantigen-binding domain and/or a second signal peptide amino-terminal tothe second antigen-binding domain, wherein the first and/or secondsignal peptides comprise the amino acid sequence of SEQ ID NO: 49. Insome embodiments, the TCRM is capable of recruiting at least oneTCR-associated signaling module selected from the group consisting ofCD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotes abTCR-CD3complex formation. In some embodiments, there is a promoter operablylinked to the 5′ end of the first nucleic acid sequence, and there isnucleic acid linker selected from the group consisting of an internalribosomal entry site (IRES) and a nucleic acid encoding a self-cleaving2A peptide (such as P2A, T2A, E2A, or F2A) linking the 3′ end of firstnucleic acid sequence to the 5′ end of the second nucleic acid sequence,wherein the first nucleic acid sequence and the second nucleic acidsequence are transcribed as a single RNA under the control of thepromoter. In some embodiments, there is a promoter operably linked tothe 5′ end of the second nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of second nucleicacid sequence to the 5′ end of the first nucleic acid sequence, whereinthe first nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, the promoter is inducible. In some embodiments, the targetantigen is a cell surface antigen. In some embodiments, the cell surfaceantigen is selected from the group consisting of a protein, acarbohydrate, and a lipid. In some embodiments, the cell surface antigenis a disease-associated antigen, such as a tumor-associated orvirally-encoded antigen. In some embodiments, the cell surface antigenis CD19. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01. In some embodiments, the effector cell is a γδ T cell. Insome embodiments, the effector cell is an αβ T cell modified to block ordecrease the expression of the TCR α and/or β chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain comprising, in orderfrom amino terminus to carboxy terminus, a first antigen-binding domainand a first TCRD comprising the amino acid sequence of SEQ ID NO: 17;and b) a second nucleic acid sequence encoding a second polypeptidechain comprising, in order from amino terminus to carboxy terminus, asecond antigen-binding domain and a second TCRD comprising the aminoacid sequence of SEQ ID NO: 18; wherein the first antigen-binding domainand the second antigen-binding domain form a Fab-like antigen-bindingmodule that specifically binds the target antigen, wherein the firstTCRD and the second TCRD form a TCRM that is capable of recruiting atleast one TCR-associated signaling module. In some embodiments, theFab-like antigen-binding module is human, humanized, chimeric,semi-synthetic, or fully synthetic. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga) at least one T cell costimulatory signaling sequence comprising (suchas consisting of) the amino acid sequence of SEQ ID NO: 70 or 71; and/orb) an epitope tag comprising (such as consisting of) the amino acidsequence of any one of SEQ ID NOs: 50-52. In some embodiments, the abTCRfurther comprises a first signal peptide amino-terminal to the firstantigen-binding domain and/or a second signal peptide amino-terminal tothe second antigen-binding domain, wherein the first and/or secondsignal peptides comprise the amino acid sequence of SEQ ID NO: 49. Insome embodiments, the TCRM is capable of recruiting at least oneTCR-associated signaling module selected from the group consisting ofCD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotes abTCR-CD3complex formation. In some embodiments, there is a promoter operablylinked to the 5′ end of the first nucleic acid sequence, and there isnucleic acid linker selected from the group consisting of an internalribosomal entry site (IRES) and a nucleic acid encoding a self-cleaving2A peptide (such as P2A, T2A, E2A, or F2A) linking the 3′ end of firstnucleic acid sequence to the 5′ end of the second nucleic acid sequence,wherein the first nucleic acid sequence and the second nucleic acidsequence are transcribed as a single RNA under the control of thepromoter. In some embodiments, there is a promoter operably linked tothe 5′ end of the second nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of second nucleicacid sequence to the 5′ end of the first nucleic acid sequence, whereinthe first nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, the promoter is inducible. In some embodiments, the targetantigen is a cell surface antigen. In some embodiments, the cell surfaceantigen is selected from the group consisting of a protein, acarbohydrate, and a lipid. In some embodiments, the cell surface antigenis a disease-associated antigen, such as a tumor-associated orvirally-encoded antigen. In some embodiments, the cell surface antigenis CD19, ROR1, ROR2, BCMA, GPRC5D, or FCRL5. In some embodiments, thetarget antigen is a surface-presented peptide/MHC complex. In someembodiments, the peptide/MHC complex comprises a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC protein. In some embodiments, thepeptide/MHC complex comprises a peptide and an MHC protein, wherein thepeptide is derived from a protein selected from the group consisting ofWT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, and PSA. In someembodiments, the MHC protein is an MHC class I protein. In someembodiments, the MHC class I protein is HLA-A. In some embodiments, theHLA-A is HLA-A02. In some embodiments, the HLA-A02 is HLA-A*02:01. Insome embodiments, the effector cell is a γδ T cell. In some embodiments,the effector cell is an αβ T cell modified to block or decrease theexpression of the TCR α and/or β chains. In some embodiments, theeffector cell is selected from the group consisting of a cytotoxic Tcell, a helper T cell, a natural killer T cell, and a suppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain comprising, in orderfrom amino terminus to carboxy terminus, a first antigen-binding domainand a first TCRD comprising the amino acid sequence of SEQ ID NO: 19;and b) a second nucleic acid sequence encoding a second polypeptidechain comprising, in order from amino terminus to carboxy terminus, asecond antigen-binding domain and a second TCRD comprising the aminoacid sequence of SEQ ID NO: 20; wherein the first antigen-binding domainand the second antigen-binding domain form a Fab-like antigen-bindingmodule that specifically binds the target antigen, wherein the firstTCRD and the second TCRD form a TCRM that is capable of recruiting atleast one TCR-associated signaling module. In some embodiments, theFab-like antigen-binding module is human, humanized, chimeric,semi-synthetic, or fully synthetic. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga) at least one T cell costimulatory signaling sequence comprising (suchas consisting of) the amino acid sequence of SEQ ID NO: 70 or 71; and/orb) an epitope tag comprising (such as consisting of) the amino acidsequence of any one of SEQ ID NOs: 50-52. In some embodiments, the abTCRfurther comprises a first signal peptide amino-terminal to the firstantigen-binding domain and/or a second signal peptide amino-terminal tothe second antigen-binding domain, wherein the first and/or secondsignal peptides comprise the amino acid sequence of SEQ ID NO: 49. Insome embodiments, the TCRM is capable of recruiting at least oneTCR-associated signaling module selected from the group consisting ofCD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotes abTCR-CD3complex formation. In some embodiments, there is a promoter operablylinked to the 5′ end of the first nucleic acid sequence, and there isnucleic acid linker selected from the group consisting of an internalribosomal entry site (IRES) and a nucleic acid encoding a self-cleaving2A peptide (such as P2A, T2A, E2A, or F2A) linking the 3′ end of firstnucleic acid sequence to the 5′ end of the second nucleic acid sequence,wherein the first nucleic acid sequence and the second nucleic acidsequence are transcribed as a single RNA under the control of thepromoter. In some embodiments, there is a promoter operably linked tothe 5′ end of the second nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of second nucleicacid sequence to the 5′ end of the first nucleic acid sequence, whereinthe first nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, the promoter is inducible. In some embodiments, the targetantigen is a cell surface antigen. In some embodiments, the cell surfaceantigen is selected from the group consisting of a protein, acarbohydrate, and a lipid. In some embodiments, the cell surface antigenis a disease-associated antigen, such as a tumor-associated orvirally-encoded antigen. In some embodiments, the cell surface antigenis CD19, ROR1, ROR2, BCMA, GPRC5D, or FCRL5. In some embodiments, thetarget antigen is a surface-presented peptide/MHC complex. In someembodiments, the peptide/MHC complex comprises a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC protein. In some embodiments, thepeptide/MHC complex comprises a peptide and an MHC protein, wherein thepeptide is derived from a protein selected from the group consisting ofWT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, and PSA. In someembodiments, the MHC protein is an MHC class I protein. In someembodiments, the MHC class I protein is HLA-A. In some embodiments, theHLA-A is HLA-A02. In some embodiments, the HLA-A02 is HLA-A*02:01. Insome embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain comprising, in orderfrom amino terminus to carboxy terminus, a first antigen-binding domainand a first TCRD comprising the amino acid sequence of SEQ ID NO: 21;and b) a second nucleic acid sequence encoding a second polypeptidechain comprising, in order from amino terminus to carboxy terminus, asecond antigen-binding domain and a second TCRD comprising the aminoacid sequence of SEQ ID NO: 22; wherein the first antigen-binding domainand the second antigen-binding domain form a Fab-like antigen-bindingmodule that specifically binds the target antigen, wherein the firstTCRD and the second TCRD form a TCRM that is capable of recruiting atleast one TCR-associated signaling module. In some embodiments, theFab-like antigen-binding module is human, humanized, chimeric,semi-synthetic, or fully synthetic. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga) at least one T cell costimulatory signaling sequence comprising (suchas consisting of) the amino acid sequence of SEQ ID NO: 70 or 71; and/orb) an epitope tag comprising (such as consisting of) the amino acidsequence of any one of SEQ ID NOs: 50-52. In some embodiments, the abTCRfurther comprises a first signal peptide amino-terminal to the firstantigen-binding domain and/or a second signal peptide amino-terminal tothe second antigen-binding domain, wherein the first and/or secondsignal peptides comprise the amino acid sequence of SEQ ID NO: 49. Insome embodiments, the TCRM is capable of recruiting at least oneTCR-associated signaling module selected from the group consisting ofCD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotes abTCR-CD3complex formation. In some embodiments, there is a promoter operablylinked to the 5′ end of the first nucleic acid sequence, and there isnucleic acid linker selected from the group consisting of an internalribosomal entry site (IRES) and a nucleic acid encoding a self-cleaving2A peptide (such as P2A, T2A, E2A, or F2A) linking the 3′ end of firstnucleic acid sequence to the 5′ end of the second nucleic acid sequence,wherein the first nucleic acid sequence and the second nucleic acidsequence are transcribed as a single RNA under the control of thepromoter. In some embodiments, there is a promoter operably linked tothe 5′ end of the second nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of second nucleicacid sequence to the 5′ end of the first nucleic acid sequence, whereinthe first nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, the promoter is inducible. In some embodiments, the targetantigen is a cell surface antigen. In some embodiments, the cell surfaceantigen is selected from the group consisting of a protein, acarbohydrate, and a lipid. In some embodiments, the cell surface antigenis a disease-associated antigen, such as a tumor-associated orvirally-encoded antigen. In some embodiments, the cell surface antigenis CD19, ROR1, ROR2, BCMA, GPRC5D, or FCRL5. In some embodiments, thetarget antigen is a surface-presented peptide/MHC complex. In someembodiments, the peptide/MHC complex comprises a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC protein. In some embodiments, thepeptide/MHC complex comprises a peptide and an MHC protein, wherein thepeptide is derived from a protein selected from the group consisting ofWT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, and PSA. In someembodiments, the MHC protein is an MHC class I protein. In someembodiments, the MHC class I protein is HLA-A. In some embodiments, theHLA-A is HLA-A02. In some embodiments, the HLA-A02 is HLA-A*02:01. Insome embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain of the abTCR comprisinga first abTCR domain comprising the amino acid sequence of SEQ ID NO: 23and b) a second nucleic acid sequence encoding a second polypeptidechain of the abTCR comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 24, wherein the first polypeptide chain isexpressed from the first nucleic acid sequence and the secondpolypeptide chain is expressed from the second nucleic acid sequence toform the abTCR, and wherein the abTCR localizes to the surface of theeffector cell. In some embodiments, there is a promoter operably linkedto the 5′ end of the first nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of first nucleic acidsequence to the 5′ end of the second nucleic acid sequence, wherein thefirst nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, there is a promoter operably linked to the 5′ end of thesecond nucleic acid sequence, and there is nucleic acid linker selectedfrom the group consisting of an internal ribosomal entry site (IRES) anda nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A,E2A, or F2A) linking the 3′ end of second nucleic acid sequence to the5′ end of the first nucleic acid sequence, wherein the first nucleicacid sequence and the second nucleic acid sequence are transcribed as asingle RNA under the control of the promoter. In some embodiments, thepromoter is inducible. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is a γδ T cell. In someembodiments, the effector cell is an αβ T cell modified to block ordecrease the expression of the TCR α and/or β chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain of the abTCR comprisinga first abTCR domain comprising the amino acid sequence of SEQ ID NO: 25and b) a second nucleic acid sequence encoding a second polypeptidechain of the abTCR comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 26, wherein the first polypeptide chain isexpressed from the first nucleic acid sequence and the secondpolypeptide chain is expressed from the second nucleic acid sequence toform the abTCR, and wherein the abTCR localizes to the surface of theeffector cell. In some embodiments, there is a promoter operably linkedto the 5′ end of the first nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of first nucleic acidsequence to the 5′ end of the second nucleic acid sequence, wherein thefirst nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, there is a promoter operably linked to the 5′ end of thesecond nucleic acid sequence, and there is nucleic acid linker selectedfrom the group consisting of an internal ribosomal entry site (IRES) anda nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A,E2A, or F2A) linking the 3′ end of second nucleic acid sequence to the5′ end of the first nucleic acid sequence, wherein the first nucleicacid sequence and the second nucleic acid sequence are transcribed as asingle RNA under the control of the promoter. In some embodiments, thepromoter is inducible. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is a γδ T cell. In someembodiments, the effector cell is an αβ T cell modified to block ordecrease the expression of the TCR α and/or β chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain of the abTCR comprisinga first abTCR domain comprising the amino acid sequence of SEQ ID NO: 27and b) a second nucleic acid sequence encoding a second polypeptidechain of the abTCR comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 28, wherein the first polypeptide chain isexpressed from the first nucleic acid sequence and the secondpolypeptide chain is expressed from the second nucleic acid sequence toform the abTCR, and wherein the abTCR localizes to the surface of theeffector cell. In some embodiments, there is a promoter operably linkedto the 5′ end of the first nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of first nucleic acidsequence to the 5′ end of the second nucleic acid sequence, wherein thefirst nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, there is a promoter operably linked to the 5′ end of thesecond nucleic acid sequence, and there is nucleic acid linker selectedfrom the group consisting of an internal ribosomal entry site (IRES) anda nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A,E2A, or F2A) linking the 3′ end of second nucleic acid sequence to the5′ end of the first nucleic acid sequence, wherein the first nucleicacid sequence and the second nucleic acid sequence are transcribed as asingle RNA under the control of the promoter. In some embodiments, thepromoter is inducible. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is a γδ T cell. In someembodiments, the effector cell is an αβ T cell modified to block ordecrease the expression of the TCR α and/or β chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain of the abTCR comprisinga first abTCR domain comprising the amino acid sequence of SEQ ID NO: 29and b) a second nucleic acid sequence encoding a second polypeptidechain of the abTCR comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 30, wherein the first polypeptide chain isexpressed from the first nucleic acid sequence and the secondpolypeptide chain is expressed from the second nucleic acid sequence toform the abTCR, and wherein the abTCR localizes to the surface of theeffector cell. In some embodiments, there is a promoter operably linkedto the 5′ end of the first nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of first nucleic acidsequence to the 5′ end of the second nucleic acid sequence, wherein thefirst nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, there is a promoter operably linked to the 5′ end of thesecond nucleic acid sequence, and there is nucleic acid linker selectedfrom the group consisting of an internal ribosomal entry site (IRES) anda nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A,E2A, or F2A) linking the 3′ end of second nucleic acid sequence to the5′ end of the first nucleic acid sequence, wherein the first nucleicacid sequence and the second nucleic acid sequence are transcribed as asingle RNA under the control of the promoter. In some embodiments, thepromoter is inducible. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain of the abTCR comprisinga first abTCR domain comprising the amino acid sequence of SEQ ID NO: 31and b) a second nucleic acid sequence encoding a second polypeptidechain of the abTCR comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 32, wherein the first polypeptide chain isexpressed from the first nucleic acid sequence and the secondpolypeptide chain is expressed from the second nucleic acid sequence toform the abTCR, and wherein the abTCR localizes to the surface of theeffector cell. In some embodiments, there is a promoter operably linkedto the 5′ end of the first nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of first nucleic acidsequence to the 5′ end of the second nucleic acid sequence, wherein thefirst nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, there is a promoter operably linked to the 5′ end of thesecond nucleic acid sequence, and there is nucleic acid linker selectedfrom the group consisting of an internal ribosomal entry site (IRES) anda nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A,E2A, or F2A) linking the 3′ end of second nucleic acid sequence to the5′ end of the first nucleic acid sequence, wherein the first nucleicacid sequence and the second nucleic acid sequence are transcribed as asingle RNA under the control of the promoter. In some embodiments, thepromoter is inducible. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain of the abTCR comprisinga first abTCR domain comprising the amino acid sequence of SEQ ID NO: 33and b) a second nucleic acid sequence encoding a second polypeptidechain of the abTCR comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 34, wherein the first polypeptide chain isexpressed from the first nucleic acid sequence and the secondpolypeptide chain is expressed from the second nucleic acid sequence toform the abTCR, and wherein the abTCR localizes to the surface of theeffector cell. In some embodiments, there is a promoter operably linkedto the 5′ end of the first nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of first nucleic acidsequence to the 5′ end of the second nucleic acid sequence, wherein thefirst nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, there is a promoter operably linked to the 5′ end of thesecond nucleic acid sequence, and there is nucleic acid linker selectedfrom the group consisting of an internal ribosomal entry site (IRES) anda nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A,E2A, or F2A) linking the 3′ end of second nucleic acid sequence to the5′ end of the first nucleic acid sequence, wherein the first nucleicacid sequence and the second nucleic acid sequence are transcribed as asingle RNA under the control of the promoter. In some embodiments, thepromoter is inducible. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain of the abTCR comprisinga first abTCR domain comprising the amino acid sequence of SEQ ID NO: 35and b) a second nucleic acid sequence encoding a second polypeptidechain of the abTCR comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 36, wherein the first polypeptide chain isexpressed from the first nucleic acid sequence and the secondpolypeptide chain is expressed from the second nucleic acid sequence toform the abTCR, and wherein the abTCR localizes to the surface of theeffector cell. In some embodiments, there is a promoter operably linkedto the 5′ end of the first nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of first nucleic acidsequence to the 5′ end of the second nucleic acid sequence, wherein thefirst nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, there is a promoter operably linked to the 5′ end of thesecond nucleic acid sequence, and there is nucleic acid linker selectedfrom the group consisting of an internal ribosomal entry site (IRES) anda nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A,E2A, or F2A) linking the 3′ end of second nucleic acid sequence to the5′ end of the first nucleic acid sequence, wherein the first nucleicacid sequence and the second nucleic acid sequence are transcribed as asingle RNA under the control of the promoter. In some embodiments, thepromoter is inducible. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain of the abTCR comprisinga first abTCR domain comprising the amino acid sequence of SEQ ID NO: 42and b) a second nucleic acid sequence encoding a second polypeptidechain of the abTCR comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 43, wherein the first polypeptide chain isexpressed from the first nucleic acid sequence and the secondpolypeptide chain is expressed from the second nucleic acid sequence toform the abTCR, and wherein the abTCR localizes to the surface of theeffector cell. In some embodiments, there is a promoter operably linkedto the 5′ end of the first nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of first nucleic acidsequence to the 5′ end of the second nucleic acid sequence, wherein thefirst nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, there is a promoter operably linked to the 5′ end of thesecond nucleic acid sequence, and there is nucleic acid linker selectedfrom the group consisting of an internal ribosomal entry site (IRES) anda nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A,E2A, or F2A) linking the 3′ end of second nucleic acid sequence to the5′ end of the first nucleic acid sequence, wherein the first nucleicacid sequence and the second nucleic acid sequence are transcribed as asingle RNA under the control of the promoter. In some embodiments, thepromoter is inducible. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain of the abTCR comprisinga first abTCR domain comprising the amino acid sequence of SEQ ID NO: 42and b) a second nucleic acid sequence encoding a second polypeptidechain of the abTCR comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 54, wherein the first polypeptide chain isexpressed from the first nucleic acid sequence and the secondpolypeptide chain is expressed from the second nucleic acid sequence toform the abTCR, and wherein the abTCR localizes to the surface of theeffector cell. In some embodiments, there is a promoter operably linkedto the 5′ end of the first nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of first nucleic acidsequence to the 5′ end of the second nucleic acid sequence, wherein thefirst nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, there is a promoter operably linked to the 5′ end of thesecond nucleic acid sequence, and there is nucleic acid linker selectedfrom the group consisting of an internal ribosomal entry site (IRES) anda nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A,E2A, or F2A) linking the 3′ end of second nucleic acid sequence to the5′ end of the first nucleic acid sequence, wherein the first nucleicacid sequence and the second nucleic acid sequence are transcribed as asingle RNA under the control of the promoter. In some embodiments, thepromoter is inducible. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain of the abTCR comprisinga first abTCR domain comprising the amino acid sequence of SEQ ID NO: 55and b) a second nucleic acid sequence encoding a second polypeptidechain of the abTCR comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 54, wherein the first polypeptide chain isexpressed from the first nucleic acid sequence and the secondpolypeptide chain is expressed from the second nucleic acid sequence toform the abTCR, and wherein the abTCR localizes to the surface of theeffector cell. In some embodiments, there is a promoter operably linkedto the 5′ end of the first nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of first nucleic acidsequence to the 5′ end of the second nucleic acid sequence, wherein thefirst nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, there is a promoter operably linked to the 5′ end of thesecond nucleic acid sequence, and there is nucleic acid linker selectedfrom the group consisting of an internal ribosomal entry site (IRES) anda nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A,E2A, or F2A) linking the 3′ end of second nucleic acid sequence to the5′ end of the first nucleic acid sequence, wherein the first nucleicacid sequence and the second nucleic acid sequence are transcribed as asingle RNA under the control of the promoter. In some embodiments, thepromoter is inducible. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided an abTCR effector cell (such as aT cell) expressing on its surface an abTCR comprising a) a first nucleicacid sequence encoding a first polypeptide chain of the abTCR comprisinga first abTCR domain comprising the amino acid sequence of SEQ ID NO: 56and b) a second nucleic acid sequence encoding a second polypeptidechain of the abTCR comprising a second abTCR domain comprising the aminoacid sequence of SEQ ID NO: 54, wherein the first polypeptide chain isexpressed from the first nucleic acid sequence and the secondpolypeptide chain is expressed from the second nucleic acid sequence toform the abTCR, and wherein the abTCR localizes to the surface of theeffector cell. In some embodiments, there is a promoter operably linkedto the 5′ end of the first nucleic acid sequence, and there is nucleicacid linker selected from the group consisting of an internal ribosomalentry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide(such as P2A, T2A, E2A, or F2A) linking the 3′ end of first nucleic acidsequence to the 5′ end of the second nucleic acid sequence, wherein thefirst nucleic acid sequence and the second nucleic acid sequence aretranscribed as a single RNA under the control of the promoter. In someembodiments, there is a promoter operably linked to the 5′ end of thesecond nucleic acid sequence, and there is nucleic acid linker selectedfrom the group consisting of an internal ribosomal entry site (IRES) anda nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A,E2A, or F2A) linking the 3′ end of second nucleic acid sequence to the5′ end of the first nucleic acid sequence, wherein the first nucleicacid sequence and the second nucleic acid sequence are transcribed as asingle RNA under the control of the promoter. In some embodiments, thepromoter is inducible. In some embodiments, the abTCR further comprisesat least one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the epitope tag comprises any one of theamino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In any of some such embodiments described herein, the abTCR effectorcell has a lower rate of chimeric receptor internalization compared to acorresponding CAR effector cell (such as an effector cell presenting onits surface a CAR comprising the antibody moiety of the abTCR, e.g., aCAR comprising an scFv comprising the antibody variable domains of theabTCR) when compared under similar conditions. For example, in someembodiments, the abTCR effector cell has a lower rate of chimericreceptor internalization compared to the corresponding CAR effector cellfollowing target antigen-dependent stimulation of the chimeric receptoreffector cells under similar conditions. In some embodiments, the abTCReffector cell has less than about 50% (such as less than about any of45, 40, 35, 30, 25, 20, 15, 10, or 5%, including any ranges betweenthese values) abTCR internalization about 90 minutes followingstimulation with the target antigen of the abTCR. In some embodiments,the abTCR effector cell is an abTCR T cell.

In any of some such embodiments described herein, an abTCR effector cellhas a lower rate and/or incidence of exhaustion compared to acorresponding CAR effector cell (such as an effector cell presenting onits surface a CAR comprising the antibody moiety of the abTCR) whencompared under similar conditions. Effector cell exhaustion can bedetermined by any means known in the art, such as by measuring theexpression level of exhaustion markers, including, without limitation,PD-1, TIM-3 and LAG-3. For example, in some embodiments, the abTCReffector cell has a lower expression level of one or more exhaustionmarkers (such as PD-1, TIM-3 or LAG-3) compared to the corresponding CAReffector cell following target antigen-dependent stimulation of thechimeric receptor effector cells under similar conditions. In someembodiments, the abTCR effector cell has a lower incidence rate of beingpositive for one or more exhaustion markers (such as PD-1, TIM-3 orLAG-3) compared to the corresponding CAR effector cell following targetantigen-dependent stimulation of the chimeric receptor effector cellsunder similar conditions. In some embodiments, the abTCR effector cellhas an incidence rate of less than about 50% (such as less than aboutany of 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1%, including anyranges between these values) for being positive for one or moreexhaustion markers (such as PD-1, TIM-3 or LAG-3) following stimulationwith the target antigen of the abTCR. In some embodiments, the abTCReffector cell is an abTCR T cell. Incidence rate can be calculated byany means known in the art, for example, by quantifying the percentageof chimeric receptor effector cells positive for an exhaustion marker ina population of chimeric receptor effector cells, wherein the percentageof cells positive for the exhaustion marker is the incidence rate.

In any of some such embodiments described herein, the abTCR effectorcell has a lower rate and/or incidence of terminal differentiationcompared to a corresponding CAR effector cell (such as an effector cellpresenting on its surface a CAR comprising the antibody moiety of theabTCR) when compared under similar conditions. Terminal differentiationcan be determined by any means known in the art, such as by measuringthe expression level of differentiation markers, including, withoutlimitation, CD28, CCR7 and granzyme B. For example, in some embodiments,the abTCR effector cell has a lower expression level of one or moreterminal differentiation markers (such as granzyme B) and/or a greaterexpression of one or more non-terminal differentiation markers (such asCD28 or CCR7) compared to the corresponding CAR effector cell undersimilar conditions. In some embodiments, the abTCR effector cell has alower incidence rate of being positive for one or more terminaldifferentiation markers (such as granzyme B) and/or a greater incidencerate of being positive for one or more non-terminal differentiationmarkers (such as CD28 or CCR7) compared to the corresponding CAReffector cell under similar conditions. In some embodiments, the abTCReffector cell has an incidence rate of less than about 50% (such as lessthan about any of 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1%,including any ranges between these values) for being positive for one ormore terminal differentiation markers (such as granzyme B) and/or anincidence rate of more than about 10% (such as more than about any of15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%,including any ranges between these values) for being positive for one ormore non-terminal differentiation markers (such as CD28 or CCR7)following stimulation with the target antigen of the abTCR. In someembodiments, the abTCR effector cell is an abTCR T cell. Incidence ratecan be calculated by any means known in the art, for example, byquantifying the percentage of chimeric receptor effector cells positivefor a terminal differentiation marker in a population of chimericreceptor effector cells, wherein the percentage of cells positive forthe terminal differentiation marker is the incidence rate.

In any of some such embodiments described herein, the abTCR effectorcell has a greater rate of proliferation compared to a corresponding CAReffector cell (such as an effector cell presenting on its surface a CARcomprising the antibody moiety of the abTCR) when compared under similarconditions. Proliferation can be determined by any means known in theart, such as by measuring dye dilution. In some embodiments, the abTCReffector cell is an abTCR T cell.

Preparation of abTCR

In some embodiments, according to any of the abTCRs described herein,the antibody moiety is a Fab-like antigen-binding module comprisingsequences from a monoclonal antibody. In some embodiments, the Fab-likeantigen-binding module comprises V_(H), C_(H)1, V_(L), and C_(L) domainsfrom the monoclonal antibody. Monoclonal antibodies can be prepared,e.g., using hybridoma methods, such as those described by Kohler andMilstein, Nature, 256:495 (1975) and Sergeeva et al., Blood,117(16):4262-4272.

In a hybridoma method, a hamster, mouse, or other appropriate hostanimal is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes can be immunized in vitro. The immunizing agent can includea polypeptide or a fusion protein of the protein of interest, or acomplex comprising at least two molecules, such as a complex comprisinga peptide and an MHC protein. Generally, peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell. See, e.g., Goding, Monoclonal Antibodies: Principles and Practice(New York: Academic Press, 1986), pp. 59-103. Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine, and human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells can be cultured in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which prevents the growth of HGPRT-deficientcells.

In some embodiments, the immortalized cell lines fuse efficiently,support stable high-level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. In some embodiments, the immortalized cell lines are murinemyeloma lines, which can be obtained, for instance, from the SalkInstitute Cell Distribution Center, San Diego, Calif. and the AmericanType Culture Collection, Manassas, Va. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies. Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al. Monoclonal Antibody Production Techniques andApplications (Marcel Dekker, Inc.: New York, 1987) pp. 51-63.

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against thepolypeptide. The binding specificity of monoclonal antibodies producedby the hybridoma cells can be determined by immunoprecipitation or by anin vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollard,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones can besub-cloned by limiting dilution procedures and grown by standardmethods. Goding, supra. Suitable culture media for this purpose include,for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells can be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the sub-clones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

In some embodiments, according to any of the abTCRs described herein,the antibody moiety is a Fab-like antigen-binding module comprisingsequences from a clone selected from an antibody moiety library (such asa phage library presenting scFv or Fab fragments). The clone may beidentified by screening combinatorial libraries for antibody fragmentswith the desired activity or activities. For example, a variety ofmethods are known in the art for generating phage display libraries andscreening such libraries for antibodies possessing the desired bindingcharacteristics. Such methods are reviewed, e.g., in Hoogenboom et al.,Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press,Totowa, N.J., 2001) and further described, e.g., in McCafferty et al.,Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Markset al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methodsin Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J.,2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al.,J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci.USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods284(1-2): 119-132(2004).

In certain phage display methods, repertoires of V_(H) and V_(L) genesare separately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self-antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

The Fab-like antigen-binding module can be prepared using phage displayto screen libraries for antibodies specific to the target antigen (suchas a peptide/MHC class I/II complex or a cell surface antigen). Thelibrary can be a human scFv phage display library having a diversity ofat least one×10⁹ (such as at least about any of 1×10⁹, 2.5×10⁹, 5×10⁹,7.5×10⁹, 1×10¹⁰, 2.5×10¹⁰, 5×10¹⁰, 7.5×10¹⁰, or 1×10¹¹) unique humanantibody fragments. In some embodiments, the library is a naïve humanlibrary constructed from DNA extracted from human PMBCs and spleens fromhealthy donors, encompassing all human heavy and light chainsubfamilies. In some embodiments, the library is a naïve human libraryconstructed from DNA extracted from PBMCs isolated from patients withvarious diseases, such as patients with autoimmune diseases, cancerpatients, and patients with infectious diseases. In some embodiments,the library is a semi-synthetic human library, wherein heavy chain CDR3is completely randomized, with all amino acids (with the exception ofcysteine) equally likely to be present at any given position (see, e.g.,Hoet, R. M. et al., Nat. Biotechnol. 23(3):344-348, 2005). In someembodiments, the heavy chain CDR3 of the semi-synthetic human libraryhas a length from about 5 to about 24 (such as about any of 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) aminoacids. In some embodiments, the library is a fully-synthetic phagedisplay library. In some embodiments, the library is a non-human phagedisplay library.

Phage clones that bind to the target antigen with high affinity can beselected by iterative binding of phage to the target antigen, which isbound to a solid support (such as, for example, beads for solutionpanning or mammalian cells for cell panning), followed by removal ofnon-bound phage and by elution of specifically bound phage. In anexample of solution panning, the target antigen can be biotinylated forimmobilization to a solid support. The biotinylated target antigen ismixed with the phage library and a solid support, such asstreptavidin-conjugated Dynabeads M-280, and then targetantigen-phage-bead complexes are isolated. The bound phage clones arethen eluted and used to infect an appropriate host cell, such as E. coliXL1-Blue, for expression and purification. In an example of cellpanning, T2 cells (a TAP-deficient, HLA-A*02:01⁺ lymphoblast cell line)loaded with an AFP peptide are mixed with the phage library, after whichthe cells are collected and the bound clones are eluted and used toinfect an appropriate host cell for expression and purification. Thepanning can be performed for multiple (such as about any of 2, 3, 4, 5,6 or more) rounds with either solution panning, cell panning, or acombination of both, to enrich for phage clones binding specifically tothe target antigen. Enriched phage clones can be tested for specificbinding to the target antigen by any methods known in the art, includingfor example ELISA and FACS.

Human and Humanized Antibody Moieties

The abTCR antibody moieties can be human or humanized. Humanized formsof non-human (e.g., murine) antibody moieties are chimericimmunoglobulins, immunoglobulin chains, or fragments thereof (such asFv, Fab, Fab′, F(ab′)₂, scFv, or other antigen-binding subsequences ofantibodies) that typically contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibody moieties include humanimmunoglobulins, immunoglobulin chains, or fragments thereof (recipientantibody) in which residues from a CDR of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibody moieties can also comprise residues that are foundneither in the recipient antibody moiety nor in the imported CDR orframework sequences. In general, the humanized antibody moiety cancomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.See, e.g., Jones et al., Nature, 321: 522-525 (1986); Riechmann et al.,Nature, 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596(1992).

Generally, a humanized antibody moiety has one or more amino acidresidues introduced into it from a source that is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.According to some embodiments, humanization can be essentially performedfollowing the method of Winter and co-workers (Jones et al., Nature,321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988);Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substitutingrodent CDRs or CDR sequences for the corresponding sequences of a humanantibody moiety. Accordingly, such “humanized” antibody moieties areantibody moieties (U.S. Pat. No. 4,816,567), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibody moieties are typically human antibody moieties in which someCDR residues and possibly some FR residues are substituted by residuesfrom analogous sites in rodent antibodies.

As an alternative to humanization, human antibody moieties can begenerated. For example, it is now possible to produce transgenic animals(e.g., mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array into such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993);U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO97/17852. Alternatively, human antibodies can be made by introducinghuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed thatclosely resembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al.,Bio/Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859(1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., NatureBiotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995).

Human antibodies may also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275) or by using varioustechniques known in the art, including phage display libraries.Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J.Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner etal. are also available for the preparation of human monoclonalantibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1): 86-95(1991).

Variants

In some embodiments, amino acid sequence variants of the antibodymoieties provided herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody moiety. Amino acid sequence variants of anantibody moiety may be prepared by introducing appropriate modificationsinto the nucleotide sequence encoding the antibody moiety, or by peptidesynthesis. Such modifications include, for example, deletions from,and/or insertions into and/or substitutions of residues within the aminoacid sequences of the antibody moiety. Any combination of deletion,insertion, and substitution can be made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics, e.g., antigen-binding.

In some embodiments, antibody moiety variants having one or more aminoacid substitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Amino acid substitutions may beintroduced into an antibody moiety of interest and the products screenedfor a desired activity, e.g., retained/improved antigen binding ordecreased immunogenicity.

Conservative substitutions are shown in Table 1 below.

TABLE 1 CONSERVATIVE SUBSTITITIONS Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln;Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C)Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala AlaHis (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe;Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K)Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile;Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met;Phe; Ala; Norleucine Leu

Amino acids may be grouped into different classes according to commonside-chain properties:

a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

c. acidic: Asp, Glu;

d. basic: His, Lys, Arg;

e. residues that influence chain orientation: Gly, Pro;

f. aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

An exemplary substitutional variant is an affinity matured antibodymoiety, which may be conveniently generated, e.g., using phagedisplay-based affinity maturation techniques. Briefly, one or more CDRresidues are mutated and the variant antibody moieties displayed onphage and screened for a particular biological activity (e.g., bindingaffinity). Alterations (e.g., substitutions) may be made in HVRs, e.g.,to improve antibody moiety affinity. Such alterations may be made in HVR“hotspots,” i.e., residues encoded by codons that undergo mutation athigh frequency during the somatic maturation process (see, e.g.,Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or specificitydetermining residues (SDRs), with the resulting variant V_(H) or V_(L)being tested for binding affinity. Affinity maturation by constructingand reselecting from secondary libraries has been described, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., (2001).)

In some embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody moiety variants with the desiredaffinity. Another method to introduce diversity involves HVR-directedapproaches, in which several HVR residues (e.g., 4-6 residues at a time)are randomized. HVR residues involved in antigen binding may bespecifically identified, e.g., using alanine scanning mutagenesis ormodeling. CDR-H3 and CDR-L3 in particular are often targeted.

In some embodiments, substitutions, insertions, or deletions may occurwithin one or more HVRs so long as such alterations do not substantiallyreduce the ability of the antibody moiety to bind antigen. For example,conservative alterations (e.g., conservative substitutions as providedherein) that do not substantially reduce binding affinity may be made inHVRs. Such alterations may be outside of HVR “hotspots” or SDRs. In someembodiments of the variant V_(H) and V_(L) sequences provided above,each HVR either is unaltered, or contains no more than one, two or threeamino acid substitutions.

A useful method for identification of residues or regions of an antibodymoiety that may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody moiety with antigen is affected. Further substitutions maybe introduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody moiety complexcan be determined to identify contact points between the antibody moietyand antigen. Such contact residues and neighboring residues may betargeted or eliminated as candidates for substitution. Variants may bescreened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody moiety with an N-terminal methionyl residue. Otherinsertional variants of the antibody moiety include the fusion to the N-or C-terminus of the antibody moiety to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody moiety.

Derivatives

In some embodiments, an abTCR according to any of the abTCRs describedherein may be further modified to contain additional nonproteinaceousmoieties that are known in the art and readily available. The moietiessuitable for derivatization of the abTCR include but are not limited towater soluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the abTCR may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the abTCR to be improved, whether the abTCRderivative will be used in a therapy under defined conditions, etc.

In some embodiments, conjugates of an abTCR and nonproteinaceous moietythat may be selectively heated by exposure to radiation are provided. Insome embodiments, the nonproteinaceous moiety is a carbon nanotube (Kamet al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). Theradiation may be of any wavelength, and includes, but is not limited to,wavelengths that do not harm ordinary cells, but which heat thenonproteinaceous moiety to a temperature at which cells proximal to theabTCR-nonproteinaceous moiety are killed.

Preparation of abTCR Effector Cells

The present invention in one aspect provides effector cells (such aslymphocytes, for example T cells) expressing an abTCR. Exemplary methodsof preparing effector cells (such as T cells) expressing the abTCRs(abTCR effector cells, such as abTCR T cells) are provided herein.

In some embodiments, an abTCR effector cell (such as an abTCR T cell)can be generated by introducing one or more nucleic acids (including forexample a lentiviral vector) encoding an abTCR (such as any of theabTCRs described herein) that specifically binds to a target antigen(such as a disease-associated antigen) into the effector cell. Theintroduction of the one or more nucleic acids into the effector cell canbe accomplished using techniques known in the art, such as thosedescribed herein for Nucleic Acids. In some embodiments, the abTCReffector cells (such as abTCR T cells) of the invention are able toreplicate in vivo, resulting in long-term persistence that can lead tosustained control of a disease associated with expression of the targetantigen (such as cancer or viral infection).

In some embodiments, the invention relates to administering agenetically modified T cell expressing an abTCR that specifically bindsto a target antigen according to any of the abTCRs described herein forthe treatment of a patient having or at risk of developing a diseaseand/or disorder associated with expression of the target antigen (alsoreferred to herein as a “target antigen-positive” or “TA-positive”disease or disorder), including, for example, cancer or viral infection,using lymphocyte infusion. In some embodiments, autologous lymphocyteinfusion is used in the treatment. Autologous PBMCs are collected from apatient in need of treatment and T cells are activated and expandedusing the methods described herein and known in the art and then infusedback into the patient.

In some embodiments, there is provided a T cell expressing an abTCR thatspecifically binds to a target antigen according to any of the abTCRsdescribed herein (also referred to herein as an “abTCR T cell”). TheabTCR T cells of the invention can undergo robust in vivo T cellexpansion and can establish target antigen-specific memory cells thatpersist at high levels for an extended amount of time in blood and bonemarrow. In some embodiments, the abTCR T cells of the invention infusedinto a patient can eliminate target antigen-presenting cells, such astarget antigen-presenting cancer or virally-infected cells, in vivo inpatients having a target antigen-associated disease. In someembodiments, the abTCR T cells of the invention infused into a patientcan eliminate target antigen-presenting cells, such as targetantigen-presenting cancer or virally-infected cells, in vivo in patientshaving a target antigen-associated disease that is refractory to atleast one conventional treatment.

Prior to expansion and genetic modification of the T cells, a source ofT cells is obtained from a subject. T cells can be obtained from anumber of sources, including peripheral blood mononuclear cells, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Insome embodiments of the present invention, any number of T cell linesavailable in the art may be used. In some embodiments of the presentinvention, T cells can be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as Ficoll™ separation. In some embodiments, cells from thecirculating blood of an individual are obtained by apheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. In some embodiments, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In some embodiments, the cells are washed with phosphate buffered saline(PBS). In some embodiments, the wash solution lacks calcium and may lackmagnesium or may lack many if not all divalent cations. As those ofordinary skill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such asCa²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other saline solutions withor without buffer. Alternatively, the undesirable components of theapheresis sample may be removed and the cells directly resuspended inculture media.

In some embodiments, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3+, CD28+, CD4+, CD8+, CD45RA⁺, and CD45RO⁺ T cells,can be further isolated by positive or negative selection techniques.For example, in some embodiments, T cells are isolated by incubationwith anti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such asDYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positiveselection of the desired T cells. In some embodiments, the time periodis about 30 minutes. In some embodiments, the time period ranges from 30minutes to 36 hours or longer (including all ranges between thesevalues). In some embodiments, the time period is at least one, 2, 3, 4,5, or 6 hours. In some embodiments, the time period is 10 to 24 hours.In some embodiments, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such as inisolating tumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmune-compromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8⁺ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells, subpopulations of T cells can be preferentially selected for oragainst at culture initiation or at other time points during theprocess. Additionally, by increasing or decreasing the ratio of anti-CD3and/or anti-CD28 antibodies on the beads or other surface,subpopulations of T cells can be preferentially selected for or againstat culture initiation or at other desired time points. The skilledartisan would recognize that multiple rounds of selection can also beused in the context of this invention. In some embodiments, it may bedesirable to perform the selection procedure and use the “unselected”cells in the activation and expansion process. “Unselected” cells canalso be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4+ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD 14, CD20, CD11b, CD 16,HLA-DR, and CD8. In some embodiments, it may be desirable to enrich foror positively select for regulatory T cells which typically expressCD4+, CD25+, CD62Lhi, GITR⁺, and FoxP3⁺. Alternatively, in someembodiments, T regulatory cells are depleted by anti-CD25 conjugatedbeads or other similar methods of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In some embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in some embodiments, aconcentration of about 2 billion cells/ml is used. In some embodiments,a concentration of about 1 billion cells/ml is used. In someembodiments, greater than about 100 million cells/ml is used. In someembodiments, a concentration of cells of about any of 10, 15, 20, 25,30, 35, 40, 45, or 50 million cells/ml is used. In some embodiments, aconcentration of cells of about any of 75, 80, 85, 90, 95, or 100million cells/ml is used. In some embodiments, a concentration of about125 or about 150 million cells/ml is used. Using high concentrations canresult in increased cell yield, cell activation, and cell expansion.Further, use of high cell concentrations allows more efficient captureof cells that may weakly express target antigens of interest, such asCD28-negative T cells, or from samples where there are many tumor cellspresent (i.e., leukemic blood, tumor tissue, etc.). Such populations ofcells may have therapeutic value and would be desirable to obtain. Forexample, using high concentration of cells allows more efficientselection of CD8⁺ T cells that normally have weaker CD28 expression.

In some embodiments of the present invention, T cells are obtained froma patient directly following treatment. In this regard, it has beenobserved that following certain cancer treatments, in particulartreatments with drugs that damage the immune system, shortly aftertreatment during the period when patients would normally be recoveringfrom the treatment, the quality of T cells obtained may be optimal orimproved for their ability to expand ex vivo. Likewise, following exvivo manipulation using the methods described herein, these cells may bein a preferred state for enhanced engraftment and in vivo expansion.Thus, it is contemplated within the context of the present invention tocollect blood cells, including T cells, dendritic cells, or other cellsof the hematopoietic lineage, during this recovery phase. Further, insome embodiments, mobilization (for example, mobilization with GM-CSF)and conditioning regimens can be used to create a condition in a subjectwherein repopulation, recirculation, regeneration, and/or expansion ofparticular cell types is favored, especially during a defined window oftime following therapy. Illustrative cell types include T cells, Bcells, dendritic cells, and other cells of the immune system.

Whether prior to or after genetic modification of the T cells to expressa desirable abTCR, the T cells can be activated and expanded generallyusing methods as described, for example, in U.S. Pat. Nos. 6,352,694;6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223;6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application PublicationNo. 20060121005.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a costimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated, such as by contact with an anti-CD3antibody, or antigen-binding fragment thereof, or an anti-CD2 antibodyimmobilized on a surface, or by contact with a protein kinase Cactivator (e.g., bryostatin) in conjunction with a calcium ionophore.For co-stimulation of an accessory molecule on the surface of the Tcells, a ligand that binds the accessory molecule is used. For example,a population of T cells can be contacted with an anti-CD3 antibody andan anti-CD28 antibody, under conditions appropriate for stimulatingproliferation of the T cells. To stimulate proliferation of either CD4+T cells or CD8⁺ T cells, an anti-CD3 antibody and an anti-CD28 antibody.Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone,Besançon, France) can be used as can other methods commonly known in theart (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al.,J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol. Meth.227(1-2):53-63, 1999).

In some of any such embodiments described herein, preparation of abTCReffector cells results in minimal or substantially no exhaustion of theabTCR effector cells. For example, in some embodiments, preparationresults in fewer than about 50% (such as fewer than about any of 45, 40,35, 30, 25, 20, 15, 10, or 5%) of the abTCR effector cells becomingexhausted. Effector cell exhaustion can be determined by any means knownin the art, including any means described herein.

In some of any such embodiments described herein, preparation of abTCReffector cells results in minimal or substantially no terminaldifferentiation of the abTCR effector cells. For example, in someembodiments, preparation results in fewer than about 50% (such as fewerthan about any of 45, 40, 35, 30, 25, 20, 15, 10, or 5%) of the abTCReffector cells becoming terminally differentiated. Effector celldifferentiation can be determined by any means known in the art,including any means described herein.

In some of any such embodiments described herein, preparation of abTCReffector cells results in minimal or substantially no internalization ofabTCRs on the abTCR effector cells. For example, in some embodiments,preparation results in less than about 50% (such as less than about anyof 45, 40, 35, 30, 25, 20, 15, 10, or 5%) of abTCRs on the abTCReffector cells becoming internalized. Internalization of abTCRs on abTCReffector cells can be determined by any means known in the art,including any means described herein.

Genetic Modification

In some embodiments, the abTCR effector cells (such as abTCR T cells) ofthe invention are generated by transducing effector cells (such as Tcells prepared by the methods described herein) with a viral vectorencoding an abTCR as described herein. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the effector cell. For a review of genetherapy procedures, see Anderson, Science 256:808-813 (1992); Nabel &Feigner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166(1993); Dillon, TIBTECH 11: 167-175 (1993); Miller, Nature 357:455-460(1992); Van Brunt, Biotechnology 6(10): 1149-1 154 (1988); Vigne,Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer &Perricaudet, British Medical Bulletin 51(1):31-44 (1995); and Yu et al.,Gene Therapy 1:13-26 (1994). In some embodiments, the viral vector is alentiviral vector, and the abTCR effector cell comprises the lentiviralvector integrated into the abTCR effector cell genome. In someembodiments, the abTCR effector cell is an abTCR T cell comprising thelentiviral vector integrated into its genome.

In some embodiments, the abTCR effector cell is a T cell modified toblock or decrease the expression of one or both of the endogenous TCRchains. For example, in some embodiments, the abTCR effector cell is anαβ T cell modified to block or decrease the expression of the TCR αand/or β chains, or the abTCR effector cell is a γδ T cell modified toblock or decrease the expression of the TCR γ and/or δ chains.Modifications of cells to disrupt gene expression include any suchtechniques known in the art, including for example RNA interference(e.g., siRNA, shRNA, miRNA), gene editing (e.g., CRISPR- or TALEN-basedgene knockout), and the like.

In some embodiments, abTCR T cells with reduced expression of one orboth of the endogenous TCR chains of the T cell are generated using theCRISPR/Cas system. For a review of the CRISPR/Cas system of geneediting, see for example Jian W & Marraffini L A, Annu. Rev. Microbiol.69, 2015; Hsu P D et al., Cell, 157(6):1262-1278, 2014; and O'Connell MR et al., Nature 516:263-266, 2014. In some embodiments, abTCR T cellswith reduced expression of one or both of the endogenous TCR chains ofthe T cell are generated using TALEN-based genome editing.

Enrichment

In some embodiments, there is provided a method of enriching aheterogeneous cell population for an abTCR effector cell according toany of the abTCR effector cells described herein.

A specific subpopulation of abTCR effector cells (such as abTCR T cells)that specifically bind to a target antigen can be enriched for bypositive selection techniques. For example, in some embodiments, abTCReffector cells (such as abTCR T cells) are enriched for by incubationwith target antigen-conjugated beads for a time period sufficient forpositive selection of the desired abTCR effector cells. In someembodiments, the time period is about 30 minutes. In some embodiments,the time period ranges from 30 minutes to 36 hours or longer (includingall ranges between these values). In some embodiments, the time periodis at least one, 2, 3, 4, 5, or 6 hours. In some embodiments, the timeperiod is 10 to 24 hours. In some embodiments, the incubation timeperiod is 24 hours. For isolation of abTCR effector cells present at lowlevels in the heterogeneous cell population, use of longer incubationtimes, such as 24 hours, can increase cell yield. Longer incubationtimes may be used to isolate abTCR effector cells in any situation wherethere are few abTCR effector cells as compared to other cell types. Theskilled artisan would recognize that multiple rounds of selection canalso be used in the context of this invention.

For isolation of a desired population of abTCR effector cells bypositive selection, the concentration of cells and surface (e.g.,particles such as beads) can be varied. In some embodiments, it may bedesirable to significantly decrease the volume in which beads and cellsare mixed together (i.e., increase the concentration of cells), toensure maximum contact of cells and beads. For example, in someembodiments, a concentration of about 2 billion cells/ml is used. Insome embodiments, a concentration of about 1 billion cells/ml is used.In some embodiments, greater than about 100 million cells/ml is used. Insome embodiments, a concentration of cells of about any of 10, 15, 20,25, 30, 35, 40, 45, or 50 million cells/ml is used. In some embodiments,a concentration of cells of about any of 75, 80, 85, 90, 95, or 100million cells/ml is used. In some embodiments, a concentration of about125 or about 150 million cells/ml is used. Using high concentrations canresult in increased cell yield, cell activation, and cell expansion.Further, use of high cell concentrations allows more efficient captureof abTCR effector cells that may weakly express the abTCR.

In some of any such embodiments described herein, enrichment results inminimal or substantially no exhaustion of the abTCR effector cells. Forexample, in some embodiments, enrichment results in fewer than about 50%(such as fewer than about any of 45, 40, 35, 30, 25, 20, 15, 10, or 5%)of the abTCR effector cells becoming exhausted. Effector cell exhaustioncan be determined by any means known in the art, including any meansdescribed herein.

In some of any such embodiments described herein, enrichment results inminimal or substantially no terminal differentiation of the abTCReffector cells. For example, in some embodiments, enrichment results infewer than about 50% (such as fewer than about any of 45, 40, 35, 30,25, 20, 15, 10, or 5%) of the abTCR effector cells becoming terminallydifferentiated. Effector cell differentiation can be determined by anymeans known in the art, including any means described herein.

In some of any such embodiments described herein, enrichment results inminimal or substantially no internalization of abTCRs on the abTCReffector cells. For example, in some embodiments, enrichment results inless than about 50% (such as less than about any of 45, 40, 35, 30, 25,20, 15, 10, or 5%) of abTCRs on the abTCR effector cells becominginternalized. Internalization of abTCRs on abTCR effector cells can bedetermined by any means known in the art, including any means describedherein.

In some of any such embodiments described herein, enrichment results inincreased proliferation of the abTCR effector cells. For example, insome embodiments, enrichment results in an increase of at least about10% (such as at least about any of 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 1000% or more) in the number of abTCR effector cellsfollowing enrichment.

Thus, in some embodiments, there is provided a method of enriching aheterogeneous cell population for abTCR effector cells expressing anabTCR that specifically binds to a target antigen comprising: a)contacting the heterogeneous cell population with a ligand comprisingthe target antigen or one or more epitopes contained therein to formcomplexes comprising the abTCR effector cell bound to the ligand; and b)separating the complexes from the heterogeneous cell population, therebygenerating a cell population enriched for the abTCR effector cells. Insome embodiments, the ligand is immobilized to a solid support. In someembodiments, the solid support is particulate (such as beads). In someembodiments, the solid support is a surface (such as the bottom of awell). In some embodiments, the ligand is labelled with a tag. In someembodiments, the tag is a fluorescent molecule, an affinity tag, or amagnetic tag. In some embodiments, the method further comprises elutingthe abTCR effector cells from the ligand and recovering the eluate.

Library Screening

To isolate candidate abTCR constructs specific for a target antigen, anabTCR library, for example cells expressing a library of nucleic acidsencoding a plurality of abTCRs, may be exposed to a ligand comprisingthe target antigen or one or more epitopes contained therein, followedby isolation of affinity members of the library that specifically bindthe ligand. In some embodiments, the ligand is immobilized on a solidsupport. In some embodiments, the support may be the surfaces of beads,microtitre plates, immunotubes, or any material known in the art usefulfor such purposes. In some embodiments, the interaction takes place insolution on tagged ligand targets (e.g. biotinylated ligand). In someembodiments, the procedure involves one or more washing steps to removeunspecific and non-reactive library members (panning). In someembodiments, to purify complexes in solution, they are captured byeither immobilization or by centrifugation. In some embodiments,affinity members are captured on a soluble biotinylated ligand, followedby immobilization of the affinity complex (affinity member and ligand)on streptavidin beads. In some embodiments, the solid support is a bead.In some embodiments, the beads include, for example, magnetic beads(e.g. from Bangs Laboratories, Polysciences inc., Dynal Biotech,Miltenyi Biotech or Quantum Magnetic), nonmagnetic beads (e.g. Pierceand Upstate technology), monodisperse beads (e.g. Dynal Biotech andMicroparticle Gmbh), and polydisperse beads (e.g. Chemagen). The use ofmagnetic beads has been described exhaustingly in literature (Uhlen, M,et al (1994) in Advances in Biomagnetic Separation, BioTechniques press,Westborough, Mass.). In some embodiments, the affinity members arepurified by positive selection. In some embodiments, the affinitymembers are purified by negative selection to remove unwanted librarymembers. In some embodiments, the affinity members are purified by bothpositive and negative selection steps.

Generally, the techniques used to prepare the library constructs will bebased on known genetic engineering techniques. In this regard, nucleicacid sequences encoding the abTCRs to be expressed in the library areincorporated into expression vectors appropriate for the type ofexpression system to be used. Appropriate expression vectors for use indisplay in cells, such as CD3+ cells, are well known and described inthe art. For example, in some embodiments, the expression vector is aviral vector, such as a lentiviral vector.

In some embodiments, there is provided a nucleic acid library comprisingsequences encoding a plurality of abTCRs according to any one of theembodiments described herein. In some embodiments, the nucleic acidlibrary comprises viral vectors encoding the plurality of abTCRs. Insome embodiments, the viral vectors are lentiviral vectors.

In some embodiments, there is provided a method of screening a nucleicacid library according to any of the embodiments described herein forsequences encoding abTCRs specific for a target antigen, comprising: a)introducing the nucleic acid library into a plurality of cells, suchthat the abTCRs are expressed on the surface of the plurality of cells;b) incubating the plurality of cells with a ligand comprising the targetantigen or one or more epitopes contained therein; c) collecting cellsbound to the ligand; and d) isolating sequences encoding the abTCRs fromcells collected in step c), thereby identifying abTCRs specific for thetarget antigen. In some embodiments, the method further comprises one ormore wash steps. In some embodiments, the one or more wash steps arecarried out between steps b) and c). In some embodiments, the pluralityof cells is a plurality of CD3+ cells. In some embodiments, the ligandis immobilized on a solid support. In some embodiments, the solidsupport is a bead. In some embodiments, collecting cells bound to theligand comprises eluting cells from the ligand bound to the solidsupport and collecting the eluate. In some embodiments, the ligand islabelled with a tag. In some embodiments, the tag is a fluorescentmolecule, an affinity tag, or a magnetic tag. In some embodiments,collecting cells bound to the ligand comprises isolating complexescomprising the cells and the labelled ligand. In some embodiments, thecells are dissociated from the complexes.

MHC Proteins

MHC class I proteins are one of two primary classes of majorhistocompatibility complex (MHC) molecules (the other being MHC classII) and are found on nearly every nucleated cell of the body. Theirfunction is to display fragments of proteins from within the cell to Tcells; healthy cells will be ignored, while cells containing foreign ormutated proteins will be attacked by the immune system. Because MHCclass I proteins present peptides derived from cytosolic proteins, thepathway of MHC class I presentation is often called the cytosolic orendogenous pathway. Class I MHC molecules bind peptides generated mainlyfrom degradation of cytosolic proteins by the proteasome. The MHCI:peptide complex is then inserted into the plasma membrane of the cell.The peptide is bound to the extracellular part of the class I MHCmolecule. Thus, the function of the class I MHC is to displayintracellular proteins to cytotoxic T cells (CTLs). However, class I MHCcan also present peptides generated from exogenous proteins, in aprocess known as cross-presentation.

MHC class I proteins consist of two polypeptide chains, α andβ2-microglobulin (β2M). The two chains are linked noncovalently viainteraction of β2M and the α3 domain. Only the α chain is polymorphicand encoded by a HLA gene, while the β2M subunit is not polymorphic andencoded by the β-2 microglobulin gene. The α3 domain is plasmamembrane-spanning and interacts with the CD8 co-receptor of T-cells. Theα3-CD8 interaction holds the MHC I molecule in place while the T cellreceptor (TCR) on the surface of the cytotoxic T cell binds its α1-α2heterodimer ligand, and checks the coupled peptide for antigenicity. Theα1 and α2 domains fold to make up a groove for peptides to bind. MHCclass I proteins bind peptides that are 8-10 amino acid in length.

MHC class II molecules are a family of molecules normally found only onantigen-presenting cells such as dendritic cells, mononuclearphagocytes, some endothelial cells, thymic epithelial cells, and Bcells. The antigens presented by class II peptides are derived fromextracellular proteins (not cytosolic as in class I); hence, the MHCclass II-dependent pathway of antigen presentation is called theendocytic or exogenous pathway. Loading of an MHC class II moleculeoccurs by phagocytosis; extracellular proteins are endocytosed, digestedin lysosomes, and the resulting epitopic peptide fragments are loadedonto MHC class II molecules prior to their migration to the cellsurface.

Like MHC class I molecules, class II molecules are also heterodimers,but in this case consist of two homogenous peptides, an α and β chain.The subdesignation α1, α2, etc. refers to separate domains within theHLA gene; each domain is usually encoded by a different exon within thegene, and some genes have further domains that encode leader sequences,transmembrane sequences, etc. Because the antigen-binding groove of MHCclass II molecules is open at both ends while the corresponding grooveon class I molecules is closed at each end, the antigens presented byMHC class II molecules are longer, generally between 15 and 24 aminoacid residues long.

The human leukocyte antigen (HLA) genes are the human versions of theMHC genes. The three major MHC class I proteins in humans are HLA-A,HLA-B, and HLA-C, while the 3 minor ones are HLA-E, HLA-F, and HLA-G.The three major MHC class II proteins involved in antigen presentationin humans are HLA-DP, HLDA-DQ, and HLA-DR, while the other MHC class IIproteins, HLA-DM and HLA-DO, are involved in the internal processing andloading of antigens. HLA-A is ranked among the genes in humans with thefastest-evolving coding sequence. As of December 2013, there were 2432known HLA-A alleles coding for 1740 active proteins and 117 nullproteins. The HLA-A gene is located on the short arm of chromosome 6 andencodes the larger, α-chain, constituent of HLA-A. Variation of HLA-Aα-chain is key to HLA function. This variation promotes geneticdiversity in the population. Since each HLA has a different affinity forpeptides of certain structures, greater variety of HLAs means greatervariety of antigens to be ‘presented’ on the cell surface, enhancing thelikelihood that a subset of the population will be resistant to anygiven foreign invader. This decreases the likelihood that a singlepathogen has the capability to wipe out the entire human population.Each individual can express up to two types of HLA-A, one from each oftheir parents. Some individuals will inherit the same HLA-A from bothparents, decreasing their individual HLA diversity; however, themajority of individuals will receive two different copies of HLA-A. Thissame pattern follows for all HLA groups. In other words, a person canonly express either one or two of the 2432 known HLA-A alleles.

All alleles receive at least a four digit classification, e.g.,HLA-A*02:12. The A signifies which HLA gene the allele belongs to. Thereare many HLA-A alleles, so that classification by serotype simplifiescategorization. The next pair of digits indicates this assignment. Forexample, HLA-A*02:02, HLA-A*02:04, and HLA-A*02:324 are all members ofthe A2 serotype (designated by the *02 prefix). This group is theprimary factor responsible for HLA compatibility. All numbers after thiscannot be determined by serotyping and are designated through genesequencing. The second set of digits indicates what HLA protein isproduced. These are assigned in order of discovery and as of December2013 there are 456 different HLA-A02 proteins known (assigned namesHLA-A*02:01 to HLA-A*02:456). The shortest possible HLA name includesboth of these details. Each extension beyond that signifies a nucleotidechange that may or may not change the protein.

In some embodiments, the Fab-like antigen-binding module specificallybinds to a complex comprising a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC class I protein, wherein the MHCclass I protein is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. In someembodiments, the MHC class I protein is HLA-A, HLA-B, or HLA-C. In someembodiments, the MHC class I protein is HLA-A. In some embodiments, theMHC class I protein is HLA-B. In some embodiments, the MHC class Iprotein is HLA-C. In some embodiments, the MHC class I protein isHLA-A01, HLA-A02, HLA-A03, HLA-A09, HLA-A10, HLA-A11, HLA-A19, HLA-A23,HLA-A24, HLA-A25, HLA-A26, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32,HLA-A33, HLA-A34, HLA-A36, HLA-A43, HLA-A66, HLA-A68, HLA-A69, HLA-A74,or HLA-A80. In some embodiments, the MHC class I protein is HLA-A02. Insome embodiments, the MHC class I protein is any one of HLA-A*02:01-555,such as HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05,HLA-A*02:06, HLA-A*02:07, HLA-A*02:08, HLA-A*02:09, HLA-A*02:10,HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:15,HLA-A*02:16, HLA-A*02:17, HLA-A*02:18, HLA-A*02:19, HLA-A*02:20,HLA-A*02:21, HLA-A*02:22, or HLA-A*02:24. In some embodiments, the MHCclass I protein is HLA-A*02:01. HLA-A*02:01 is expressed in 39-46% ofall Caucasians, and therefore represents a suitable choice of MHC classI protein for use in the present invention.

In some embodiments, the Fab-like antigen-binding module specificallybinds to a complex comprising a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC class II protein, wherein the MHCclass II protein is HLA-DP, HLA-DQ, or HLA-DR. In some embodiments, theMHC class II protein is HLA-DP. In some embodiments, the MHC class IIprotein is HLA-DQ. In some embodiments, the MHC class II protein isHLA-DR.

Peptides suitable for use in generating Fab-like antigen-binding modulescan be determined, for example, based on the presence of HLA (such asHLA-A*02:01) binding motifs and cleavage sites for proteasomes andimmune-proteasomes using computer prediction models known to those ofskill in the art. For predicting MHC binding sites, such models include,but are not limited to, ProPred1 (described in more detail in Singh andRaghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS17(12):1236-1237, 2001), and SYFPEITHI (see Schuler et al. SYFPEITHI,Database for Searching and T-Cell Epitope Prediction. inImmunoinformatics Methods in Molecular Biology, vol 409(1): 75-93,2007).

Once appropriate peptides have been identified, peptide synthesis may bedone in accordance with protocols well known to those of skill in theart. Because of their relatively small size, the peptides of theinvention may be directly synthesized in solution or on a solid supportin accordance with conventional peptide synthesis techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols. The synthesis of peptides in solutionphase has become a well-established procedure for large-scale productionof synthetic peptides and as such is a suitable alternative method forpreparing the peptides of the invention (See for example, Solid PhasePeptide Synthesis by John Morrow Stewart and Martin et al. Applicationof Almez-mediated Amidation Reactions to Solution Phase PeptideSynthesis, Tetrahedron Letters Vol. 39, pages 1517-1520, 1998).

Pharmaceutical Compositions

Also provided herein are compositions (such as pharmaceuticalcompositions, also referred to herein as formulations) comprising anabTCR according to any of the embodiments described herein, a nucleicacid encoding an abTCR according to any of the embodiments describedherein, or an abTCR effector cell according to any of the embodimentsdescribed herein. In some embodiments, the composition is an abTCReffector cell composition (such as a pharmaceutical composition)comprising an effector cell (such as a T cell) presenting on its surfacean abTCR according to any of the abTCRs described herein. In someembodiments, the abTCR effector cell composition is a pharmaceuticalcomposition.

The composition may comprise a homogenous cell population comprisingabTCR effector cells of the same cell type and expressing the sameabTCR, or a heterogeneous cell population comprising a plurality ofabTCR effector cell populations comprising abTCR effector cells ofdifferent cell types and/or expressing different abTCRs. The compositionmay further comprise cells that are not abTCR effector cells.

Thus, in some embodiments, there is provided an abTCR effector cellcomposition comprising a homogeneous cell population of abTCR effectorcells (such as abTCR T cells) of the same cell type and expressing thesame abTCR. In some embodiments, the abTCR effector cell is a T cell. Insome embodiments, the abTCR effector cell is selected from the groupconsisting of a cytotoxic T cell, a helper T cell, a natural killer Tcell, and a suppressor T cell. In some embodiments, the abTCR effectorcell composition is a pharmaceutical composition.

In some embodiments, there is provided an abTCR effector cellcomposition comprising a heterogeneous cell population comprising aplurality of abTCR effector cell populations comprising abTCR effectorcells of different cell types and/or expressing different abTCRs. Insome embodiments, the abTCR effector cells are T cells. In someembodiments, each population of abTCR effector cells is of a cell typeselected from the group consisting of cytotoxic T cells, helper T cells,natural killer T cells, and suppressor T cells. In some embodiments, allof the abTCR effector cells in the composition are of the same cell type(e.g., all of the abTCR effector cells are cytotoxic T cells). In someembodiments, at least one population of abTCR effector cells is of adifferent cell type than the others (e.g., one population of abTCReffector cells consists of cytotoxic T cells and the other populationsof abTCR effector cells consist of natural killer T cells). In someembodiments, each population of abTCR effector cells expresses the sameabTCR. In some embodiments, at least one population of abTCR effectorcells expresses a different abTCR than the others. In some embodiments,each population of abTCR effector cells expresses a different abTCR thanthe others. In some embodiments, each population of abTCR effector cellsexpresses an abTCR that specifically binds to the same target antigen.In some embodiments, at least one population of abTCR effector cellsexpresses an abTCR that specifically binds to a different target antigenthan the others (e.g., one population of abTCR effector cellsspecifically binds to a pMHC complex and the other populations of abTCReffector cells specifically bind to a cell surface receptor). In someembodiments, where at least one population of abTCR effector cellsexpresses an abTCR that specifically binds to a different targetantigen, each population of abTCR effector cells expresses an abTCR thatspecifically binds to a target antigen associated with the same diseaseor disorder (e.g., each of the target antigens are associated with acancer, such as breast cancer). In some embodiments, the abTCR effectorcell composition is a pharmaceutical composition.

Thus, in some embodiments, there is provided an abTCR effector cellcomposition comprising a plurality of abTCR effector cell populationsaccording to any of the embodiments described herein, wherein all of theabTCR effector cells in the composition are of the same cell type (e.g.,all of the abTCR effector cells are cytotoxic T cells), and wherein eachpopulation of abTCR effector cells expresses a different abTCR than theothers. In some embodiments, the abTCR effector cells are T cells. Insome embodiments, the abTCR effector cells are selected from the groupconsisting of cytotoxic T cells, helper T cells, natural killer T cells,and suppressor T cells. In some embodiments, each population of abTCReffector cells expresses an abTCR that specifically binds to the sametarget antigen. In some embodiments, at least one population of abTCReffector cells expresses an abTCR that specifically binds to a differenttarget antigen than the others (e.g., one population of abTCR effectorcells specifically binds to a pMHC complex and the other populations ofabTCR effector cells specifically bind to a cell surface receptor). Insome embodiments, where at least one population of abTCR effector cellsexpresses an abTCR that specifically binds to a different targetantigen, each population of abTCR effector cells expresses an abTCR thatspecifically binds to a target antigen associated with the same diseaseor disorder (e.g., each of the target antigens are associated with acancer, such as breast cancer). In some embodiments, the abTCR effectorcell composition is a pharmaceutical composition.

In some embodiments, there is provided a composition comprising aplurality of abTCR effector cell populations according to any of theembodiments described herein, wherein at least one population of abTCReffector cells is of a different cell type than the others. In someembodiments, all of the populations of abTCR effector cells are ofdifferent cell types. In some embodiments, the abTCR effector cells areT cells. In some embodiments, each population of abTCR effector cells isof a cell type selected from the group consisting of cytotoxic T cells,helper T cells, natural killer T cells, and suppressor T cells. In someembodiments, each population of abTCR effector cells expresses the sameabTCR. In some embodiments, at least one population of abTCR effectorcells expresses a different abTCR than the others. In some embodiments,each population of abTCR effector cells expresses a different abTCR thanthe others. In some embodiments, each population of abTCR effector cellsexpresses an abTCR that specifically binds to the same target antigen.In some embodiments, at least one population of abTCR effector cellsexpresses an abTCR that specifically binds to a different target antigenthan the others (e.g., one population of abTCR effector cellsspecifically binds to a pMHC complex and the other populations of abTCReffector cells specifically bind to a cell surface receptor). In someembodiments, where at least one population of abTCR effector cellsexpresses an abTCR that specifically binds to a different targetantigen, each population of abTCR effector cells expresses an abTCR thatspecifically binds to a target antigen associated with the same diseaseor disorder (e.g., each of the target antigens are associated with acancer, such as breast cancer). In some embodiments, the abTCR effectorcell composition is a pharmaceutical composition.

At various points during preparation of a composition, it can benecessary or beneficial to cryopreserve a cell. The terms“frozen/freezing” and “cryopreserved/cryopreserving” can be usedinterchangeably. Freezing includes freeze drying.

As is understood by one of ordinary skill in the art, the freezing ofcells can be destructive (see Mazur, P., 1977, Cryobiology 14:251-272)but there are numerous procedures available to prevent such damage. Forexample, damage can be avoided by (a) use of a cryoprotective agent, (b)control of the freezing rate, and/or (c) storage at a temperaturesufficiently low to minimize degradative reactions. Exemplarycryoprotective agents include dimethyl sulfoxide (DMSO) (Lovelock andBishop, 1959, Nature 183:1394-1395; Ashwood-Smith, 1961, Nature190:1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, 1960, Ann. N.Y.Acad. Sci. 85:576), polyethylene glycol (Sloviter and Ravdin, 1962,Nature 196:548), albumin, dextran, sucrose, ethylene glycol,i-erythritol, D-ribitol, D-mannitol (Rowe et al., 1962, Fed. Proc.21:157), D-sorbitol, i-inositol, D-lactose, choline chloride (Bender etal., 1960, J. Appl. Physiol. 15:520), amino acids (Phan The Tran andBender, 1960, Exp. Cell Res. 20:651), methanol, acetamide, glycerolmonoacetate (Lovelock, 1954, Biochem. J. 56:265), and inorganic salts(Phan The Tran and Bender, 1960, Proc. Soc. Exp. Biol. Med. 104:388;Phan The Tran and Bender, 1961, in Radiobiology, Proceedings of theThird Australian Conference on Radiobiology, llbery ed., Butterworth,London, p. 59). In particular embodiments, DMSO can be used. Addition ofplasma (e.g., to a concentration of 20-25%) can augment the protectiveeffects of DMSO. After addition of DMSO, cells can be kept at 0° C.until freezing, because DMSO concentrations of 1% can be toxic attemperatures above 4° C.

In the cryopreservation of cells, slow controlled cooling rates can becritical and different cryoprotective agents (Rapatz et al., 1968,Cryobiology 5(1): 18-25) and different cell types have different optimalcooling rates (see e.g., Rowe and Rinfret, 1962, Blood 20:636; Rowe,1966, Cryobiology 3(1):12-18; Lewis, et al., 1967, Transfusion7(1):17-32; and Mazur, 1970, Science 168:939-949 for effects of coolingvelocity on survival of stem cells and on their transplantationpotential). The heat of fusion phase where water turns to ice should beminimal. The cooling procedure can be carried out by use of, e.g., aprogrammable freezing device or a methanol bath procedure. Programmablefreezing apparatuses allow determination of optimal cooling rates andfacilitate standard reproducible cooling.

In particular embodiments, DMSO-treated cells can be pre-cooled on iceand transferred to a tray containing chilled methanol which is placed,in turn, in a mechanical refrigerator (e.g., Harris or Revco) at −80° C.Thermocouple measurements of the methanol bath and the samples indicatea cooling rate of 1° to 3° C./minute can be preferred. After at leasttwo hours, the specimens can have reached a temperature of −80° C. andcan be placed directly into liquid nitrogen (−196° C.).

After thorough freezing, the cells can be rapidly transferred to along-term cryogenic storage vessel. In a preferred embodiment, samplescan be cryogenically stored in liquid nitrogen (−196° C.) or vapor (−1°C.). Such storage is facilitated by the availability of highly efficientliquid nitrogen refrigerators.

Further considerations and procedures for the manipulation,cryopreservation, and long-term storage of cells, can be found in thefollowing exemplary references: U.S. Pat. Nos. 4,199,022; 3,753,357; and4,559,298; Gorin, 1986, Clinics In Haematology 15(1):19-48; Bone-MarrowConservation, Culture and Transplantation, Proceedings of a Panel,Moscow, Jul. 22-26, 1968, International Atomic Energy Agency, Vienna,pp. 107-186; Livesey and Linner, 1987, Nature 327:255; Linner et al.,1986, J. Histochem. Cytochem. 34(9):1 123-1 135; Simione, 1992, J.Parenter. Sci. Technol. 46(6):226-32).

Following cryopreservation, frozen cells can be thawed for use inaccordance with methods known to those of ordinary skill in the art.Frozen cells are preferably thawed quickly and chilled immediately uponthawing. In particular embodiments, the vial containing the frozen cellscan be immersed up to its neck in a warm water bath; gentle rotationwill ensure mixing of the cell suspension as it thaws and increase heattransfer from the warm water to the internal ice mass. As soon as theice has completely melted, the vial can be immediately placed on ice.

In particular embodiments, methods can be used to prevent cellularclumping during thawing. Exemplary methods include: the addition beforeand/or after freezing of DNase (Spitzer et al., 1980, Cancer45:3075-3085), low molecular weight dextran and citrate, hydroxyethylstarch (Stiff et al., 1983, Cryobiology 20:17-24), etc. [0162] As isunderstood by one of ordinary skill in the art, if a cryoprotectiveagent that is toxic to humans is used, it should be removed prior totherapeutic use. DMSO has no serious toxicity.

Exemplary carriers and modes of administration of cells are described atpages 14-15 of U.S. Patent Publication No. 2010/0183564. Additionalpharmaceutical carriers are described in Remington: The Science andPractice of Pharmacy, 21 st Edition, David B. Troy, ed., LippicottWilliams & Wilkins (2005).

In particular embodiments, cells can be harvested from a culture medium,and washed and concentrated into a carrier in atherapeutically-effective amount. Exemplary carriers include saline,buffered saline, physiological saline, water, Hanks' solution, Ringer'ssolution, Nonnosol-R (Abbott Labs), Plasma-Lyte A(R) (BaxterLaboratories, Inc., Morton Grove, Ill.), glycerol, ethanol, andcombinations thereof.

In particular embodiments, carriers can be supplemented with human serumalbumin (HSA) or other human serum components or fetal bovine serum. Inparticular embodiments, a carrier for infusion includes buffered salinewith 5% HAS or dextrose. Additional isotonic agents include polyhydricsugar alcohols including trihydric or higher sugar alcohols, such asglycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

Carriers can include buffering agents, such as citrate buffers,succinate buffers, tartrate buffers, fumarate buffers, gluconatebuffers, oxalate buffers, lactate buffers, acetate buffers, phosphatebuffers, histidine buffers, and/or trimethylamine salts.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which helps to prevent celladherence to container walls. Typical stabilizers can include polyhydricsugar alcohols; amino acids, such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, and threonine; organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, suchas inositol; PEG; amino acid polymers; sulfur-containing reducingagents, such as urea, glutathione, thioctic acid, sodium thioglycolate,thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; lowmolecular weight polypeptides (i.e., <10 residues); proteins such asHSA, bovine serum albumin, gelatin or immunoglobulins; hydrophilicpolymers such as polyvinylpyrrolidone; monosaccharides such as xylose,mannose, fructose and glucose; disaccharides such as lactose, maltoseand sucrose; trisaccharides such as raffinose, and polysaccharides suchas dextran.

Where necessary or beneficial, compositions can include a localanesthetic such as lidocaine to ease pain at a site of injection.

Exemplary preservatives include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalkonium halides, hexamethonium chloride, alkyl parabenssuch as methyl or propyl paraben, catechol, resorcinol, cyclohexanol,and 3-pentanol.

Therapeutically effective amounts of cells within compositions can begreater than 10² cells, greater than 10³ cells, greater than 10⁴ cells,greater than 10⁵ cells, greater than 10⁶ cells, greater than 10⁷ cells,greater than 10⁸ cells, greater than 10⁹ cells, greater than 10¹⁰ cells,or greater than 10¹¹ cells.

In compositions and formulations disclosed herein, cells are generallyin a volume of a liter or less, 500 ml or less, 250 ml or less or 100 mlor less. Hence the density of administered cells is typically greaterthan 10⁴ cells/ml, 10⁷ cells/ml or 10⁸ cells/ml.

Also provided herein are abTCR nucleic acid compositions (such aspharmaceutical compositions, also referred to herein as formulations)comprising any of the nucleic acids encoding an abTCR described herein.In some embodiments, the abTCR nucleic acid composition is apharmaceutical composition. In some embodiments, the abTCR nucleic acidcomposition further comprises any of an isotonizing agent, an excipient,a diluent, a thickener, a stabilizer, a buffer, and/or a preservative;and/or an aqueous vehicle, such as purified water, an aqueous sugarsolution, a buffer solution, physiological saline, an aqueous polymersolution, or RNase free water. Tsubunit ishe amounts of such additivesand aqueous vehicles to be added can be suitably selected according tothe form of use of the abTCR nucleic acid composition.

The compositions and formulations disclosed herein can be prepared foradministration by, for example, injection, infusion, perfusion, orlavage. The compositions and formulations can further be formulated forbone marrow, intravenous, intradermal, intraarterial, intranodal,intralymphatic, intraperitoneal, intralesional, intraprostatic,intravaginal, intrarectal, topical, intrathecal, intratumoral,intramuscular, intravesicular, and/or subcutaneous injection.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by, e.g., filtration through sterilefiltration membranes.

Methods of Treatment Using abTCRs

The abTCRs and/or compositions of the invention can be administered toindividuals (e.g., mammals such as humans) to treat a disease and/ordisorder associated with target antigen (TA) expression (also referredto herein as a “target-antigen positive” or “TA-positive” disease ordisorder), including, for example, cancer and infectious disease (suchas viral infection). The present application thus in some embodimentsprovides a method for treating a target antigen-positive disease (suchas cancer or viral infection) in an individual comprising administeringto the individual an effective amount of a composition (such as apharmaceutical composition) comprising an abTCR comprising an antibodymoiety, such as any one of the abTCRs described herein. In someembodiments, the composition further comprises a cell (such as aneffector cell) associated with the abTCR. In some embodiments, thecancer is selected, for example, from the group consisting ofadrenocortical carcinoma, bladder cancer, breast cancer, cervicalcancer, cholangiocarcinoma, colorectal cancers, esophageal cancer,glioblastoma, glioma, hepatocellular carcinoma, head and neck cancer,kidney cancer, lung cancer, melanoma, mesothelioma, multiple myeloma,pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma,ovarian cancer, prostate cancer, sarcoma, stomach cancer, uterine cancerand thyroid cancer. In some embodiments, the viral infection is causedby a virus selected, for example, from the group consisting ofCytomegalovirus (CMV), Epstein-Barr Virus (EBV), Hepatitis B Virus(HBV), Kaposi's Sarcoma associated herpesvirus (KSHV), Humanpapillomavirus (HPV), Molluscum contagiosum virus (MCV), Human T cellleukemia virus 1 (HTLV-1), HIV (Human immunodeficiency virus), andHepatitis C Virus (HCV).

For example, in some embodiments, there is provided a method of treatinga target antigen-associated disease (such as cancer or viral infection)in an individual in need thereof comprising administering to theindividual an effective amount of a composition comprising effectorcells (such as T cells or natural killer cells) presenting on theirsurface an abTCR (such as an isolated abTCR) comprising a) a Fab-likeantigen-binding module that specifically binds to the target antigen,and b) a TCRM capable of recruiting at least one TCR-associatedsignaling module. In some embodiments, the Fab-like antigen-bindingmodule comprises a V_(H) antibody domain, a C_(H)1 antibody domain, aV_(L) antibody domain, and a C_(L) antibody domain. In some embodiments,the Fab-like antigen-binding module is human, humanized, chimeric,semi-synthetic, or fully synthetic. In some embodiments, the TCRMcomprises the transmembrane domains of a TCR, such as an αβTCR or aγδTCR. In some embodiments, the TCRM further comprises the connectingpeptides or fragments thereof of the TCR. In some embodiments, the TCRMfurther comprises at least one portion of an extracellular domain of theTCR. In some embodiments, the abTCR further comprises at least oneintracellular domain. In some embodiments, the at least oneintracellular domain comprises any of a sequence from an intracellulardomain of the TCR, a co-stimulatory intracellular signaling sequence, anepitope tag, or a combination thereof. In some embodiments, the abTCRfurther comprises at least one disulfide bond. In some embodiments, theFab-like antigen binding module comprises a disulfide bond and/or theTCRM comprises a disulfide bond. In some embodiments, the Fab-likeantigen binding module comprises a disulfide bond between a residue inthe C_(H)1 domain and a residue in the C_(L) domain and/or the TCRMcomprises a disulfide bond between a residue in the first connectingpeptide and a residue in the second connecting peptide. In someembodiments, the TCRM is capable of recruiting at least oneTCR-associated signaling module selected from the group consisting ofCD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotes abTCR-CD3complex formation. In some embodiments, there is a peptide linkerbetween the Fab-like antigen-binding module and the TCRM. In someembodiments, the target antigen is a cell surface antigen. In someembodiments, the cell surface antigen is selected from the groupconsisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the method results in minimal or substantially noexhaustion of the abTCR effector cells. In some embodiments, the methodresults in minimal or substantially no terminal differentiation of theabTCR effector cells. In some embodiments, the method results in minimalor substantially no internalization of abTCRs on the abTCR effectorcells. In some embodiments, the method results in increasedproliferation of the abTCR effector cells.

In some embodiments, there is provided a method of treating a targetantigen-associated disease (such as cancer or viral infection) in anindividual in need thereof comprising administering to the individual aneffective amount of a composition comprising effector cells (such as Tcells or natural killer cells) presenting on their surface an abTCR(such as an isolated abTCR) comprising a) an Fv-like antigen-bindingmodule that specifically binds to a target antigen, and b) a TCRMcapable of recruiting at least one TCR-associated signaling module,wherein the target antigen is a peptide/MHC complex. In someembodiments, the Fv-like antigen-binding module comprises a V_(H)antibody domain and a V_(L) antibody domain. In some embodiments, thereis a first peptide linker fused to the C-terminus of the V_(L) antibodydomain and/or a second peptide linker fused to the C-terminus of theV_(H) antibody domain. In some embodiments, the first and second peptidelinkers are capable of binding to one another. In some embodiments, thefirst and/or second peptide linkers are derived from immunoglobulinheavy and/or light chain constant regions. In some embodiments, thefirst and/or second peptide linkers are derived from TCR subunitconstant regions. For example, in some embodiments, the first and/orsecond peptide linkers are derived from a) TCR α and β subunit constantdomains; or b) TCR γ and δ subunit constant domains. In someembodiments, the first and/or second peptide linkers are synthetic. Insome embodiments, the Fv-like antigen-binding module is human,humanized, chimeric, semi-synthetic, or fully synthetic. In someembodiments, the TCRM comprises the transmembrane domains of a TCR, suchas an αβTCR or a γδTCR. In some embodiments, the TCRM further comprisesthe connecting peptides or fragments thereof of a TCR, such as an αβTCRor a γδTCR. In some embodiments, the transmembrane domains and theconnecting peptides are derived from an αβTCR or a γδTCR. In someembodiments, the transmembrane domains are derived from an αβTCR and theconnecting peptides are derived from a γδTCR, or the transmembranedomains are derived from a γδTCR and the connecting peptides are derivedfrom an αβTCR. In some embodiments, the TCRM further comprises at leastone portion of an extracellular domain of the TCR. In some embodiments,the TCRM further comprises at least one TCR intracellular domaincomprising a sequence from an intracellular domain of the TCR. In someembodiments, the TCRM comprises fragments of the TCR subunits. In someembodiments, the abTCR further comprises at least one accessoryintracellular domain comprising a T cell costimulatory signalingsequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD40)and/or an epitope tag (such as HA, FLAG, or myc). In some embodiments,the abTCR further comprises a first signal peptide amino-terminal to thefirst antigen-binding domain and/or a second signal peptideamino-terminal to the second antigen-binding domain. In someembodiments, the abTCR further comprises at least one disulfide bond. Insome embodiments, the first and/or second peptide linkers comprise adisulfide bond and/or the TCRM comprises a disulfide bond. In someembodiments, the TCRM comprises a disulfide bond between a residue inthe first connecting peptide and a residue in the second connectingpeptide. In some embodiments, the TCRM is capable of recruiting at leastone TCR-associated signaling module selected from the group consistingof CD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotesabTCR-CD3 complex formation. In some embodiments, the target antigenpeptide/MHC complex comprises a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC protein. In some embodiments, thepeptide/MHC complex comprises a peptide and an MHC protein, wherein thepeptide is derived from a protein selected from the group consisting ofWT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, and PSA. In someembodiments, the MHC protein is an MHC class I protein. In someembodiments, the MHC class I protein is HLA-A. In some embodiments, theHLA-A is HLA-A02. In some embodiments, the HLA-A02 is HLA-A*02:01. Insome embodiments, the method results in minimal or substantially noexhaustion of the abTCR effector cells. In some embodiments, the methodresults in minimal or substantially no terminal differentiation of theabTCR effector cells. In some embodiments, the method results in minimalor substantially no internalization of abTCRs on the abTCR effectorcells. In some embodiments, the method results in increasedproliferation of the abTCR effector cells.

In some embodiments, there is provided a method of treating a targetantigen-associated disease (such as cancer or viral infection) in anindividual in need thereof comprising administering to the individual aneffective amount of a composition comprising effector cells (such as Tcells or natural killer cells) presenting on their surface an abTCR thatspecifically recognizes the target antigen comprising a) a firstpolypeptide chain comprising a first antigen-binding domain comprisingV_(H) and C_(H)1 antibody domains and a first TCRD comprising thetransmembrane domain of a first TCR subunit; and b) a second polypeptidechain comprising a second antigen-binding domain comprising V_(L) andC_(L) antibody domains and a second TCRD comprising the transmembranedomain of a second TCR subunit, wherein the V_(H) and C_(H)1 domains ofthe first antigen-binding domain and the V_(L) and C_(L) domains of thesecond antigen-binding domain form a Fab-like antigen-binding modulethat specifically binds the target antigen, wherein the first TCRD andthe second TCRD form a TCRM that is capable of recruiting at least oneTCR-associated signaling module. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the first TCR subunit is a TCR αchain, and the second TCR subunit is a TCR β chain. In some embodiments,the first TCR subunit is a TCR β chain, and the second TCR subunit is aTCR α chain. In some embodiments, the first TCR subunit is a TCR γchain, and the second TCR subunit is a TCR δ chain. In some embodiments,the first TCR subunit is a TCR δ chain, and the second TCR subunit is aTCR γ chain. In some embodiments, the first TCRD further comprises theconnecting peptide or a fragment thereof of the first TCR subunit and/orthe second TCRD further comprises the connecting peptide or a fragmentthereof of the second TCR subunit. In some embodiments, the first TCRDfurther comprises a portion of the extracellular domain of the first TCRsubunit and/or the second TCRD further comprises a portion of theextracellular domain of the second TCR subunit. In some embodiments, thefirst TCRD further comprises a first TCR intracellular domain and/or thesecond TCRD further comprises a second TCR intracellular domain. In someembodiments, the first TCR intracellular domain comprises a sequencefrom the intracellular domain of the first TCR subunit and/or the secondTCR intracellular domain comprises a sequence from the intracellulardomain of the second TCR subunit. In some embodiments, the abTCR furthercomprises a first signal peptide amino-terminal to the firstantigen-binding domain and/or a second signal peptide amino-terminal tothe second antigen-binding domain. In some embodiments, the TCRM iscapable of recruiting at least one TCR-associated signaling moduleselected from the group consisting of CD3δε, CD3γε, and ζζ. In someembodiments, the TCRM promotes abTCR-CD3 complex formation. In someembodiments, there is a first peptide linker between the firstantigen-binding domain and the first TCRD and/or a second peptide linkerbetween the second antigen-binding domain and the second TCRD. In someembodiments, the first and second polypeptide chains are linked, such asby a covalent linkage (e.g., peptide or other chemical linkage) ornon-covalent linkage. In some embodiments, the first polypeptide chainand the second polypeptide chain are linked via a) a disulfide bondbetween a residue in the connecting peptide of the first TCRD and aresidue in the connecting peptide of the second TCRD; and/or b) adisulfide bond between a residue in the C_(H)1 antibody domain in thefirst antigen-binding domain and a residue in the C_(L) antibody domainin the second antigen-binding domain. In some embodiments, the targetantigen is a cell surface antigen. In some embodiments, the cell surfaceantigen is selected from the group consisting of a protein, acarbohydrate, and a lipid. In some embodiments, the cell surface antigenis a disease-associated antigen, such as a tumor-associated orvirally-encoded antigen. In some embodiments, the cell surface antigenis CD19, ROR1, ROR2, BCMA, GPRC5D, or FCRL5. In some embodiments, thetarget antigen is a surface-presented peptide/MHC complex. In someembodiments, the peptide/MHC complex comprises a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC protein. In some embodiments, thepeptide/MHC complex comprises a peptide and an MHC protein, wherein thepeptide is derived from a protein selected from the group consisting ofWT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, and PSA.

In some embodiments, there is provided a method of treating a targetantigen-associated disease (such as cancer or viral infection) in anindividual in need thereof comprising administering to the individual aneffective amount of a composition comprising effector cells (such as Tcells or natural killer cells) presenting on their surface an abTCR thatspecifically recognizes the target antigen comprising a) a firstpolypeptide chain comprising a first antigen-binding domain comprisingV_(H) and C_(H)1 antibody domains and a first TCRD comprising thetransmembrane domain of a TCR α chain; and b) a second polypeptide chaincomprising a second antigen-binding domain comprising V_(L) and C_(L)antibody domains and a second TCRD comprising the transmembrane domainof a TCR β chain, wherein the V_(H) and C_(H)1 domains of the firstantigen-binding domain and the V_(L) and C_(L) domains of the secondantigen-binding domain form a Fab-like antigen-binding module thatspecifically binds to the cell surface antigen, wherein the first TCRDand the second TCRD form a TCRM that is capable of recruiting at leastone TCR-associated signaling module. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the first TCRD further comprisesthe connecting peptide or a fragment thereof of the TCR α chain and/orthe second TCRD further comprises the connecting peptide or a fragmentthereof of the TCR β chain. In some embodiments, the first TCRD furthercomprises a portion of the extracellular domain of the TCR α chainand/or the second TCRD further comprises a portion of the extracellulardomain of the TCR β chain. In some embodiments, the first TCRD furthercomprises a first TCR intracellular domain and/or the second TCRDfurther comprises a second TCR intracellular domain. In someembodiments, the first TCR intracellular domain comprises a sequencefrom the intracellular domain of the TCR α chain and/or the second TCRintracellular domain comprises a sequence from the intracellular domainof the TCR β chain. In some embodiments, the abTCR further comprises atleast one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the abTCR further comprises a first signalpeptide amino-terminal to the first antigen-binding domain and/or asecond signal peptide amino-terminal to the second antigen-bindingdomain. In some embodiments, the TCRM is capable of recruiting at leastone TCR-associated signaling module selected from the group consistingof CD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotesabTCR-CD3 complex formation. In some embodiments, there is a firstpeptide linker between the first antigen-binding domain and the firstTCRD and/or a second peptide linker between the second antigen-bindingdomain and the second TCRD. In some embodiments, the first and secondpolypeptide chains are linked, such as by a covalent linkage (e.g.,peptide or other chemical linkage) or non-covalent linkage. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between a residuein the C_(H)1 antibody domain in the first antigen-binding domain and aresidue in the C_(L) antibody domain in the second antigen-bindingdomain. In some embodiments, the target antigen is a cell surfaceantigen. In some embodiments, the cell surface antigen is selected fromthe group consisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01. In some embodiments, the effector cell is a γδ T cell. Insome embodiments, the effector cell is an αβ T cell modified to block ordecrease the expression of the TCR α and/or β chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating a targetantigen-associated disease (such as cancer or viral infection) in anindividual in need thereof comprising administering to the individual aneffective amount of a composition comprising effector cells (such as Tcells or natural killer cells) presenting on their surface an abTCR thatspecifically recognizes the target antigen comprising a) a firstpolypeptide chain comprising a first antigen-binding domain comprisingV_(H) and C_(H)1 antibody domains and a first TCRD comprising thetransmembrane domain of a TCR β chain; and b) a second polypeptide chaincomprising a second antigen-binding domain comprising V_(L) and C_(L)antibody domains and a second TCRD comprising the transmembrane domainof a TCR α chain, wherein the V_(H) and C_(H)1 domains of the firstantigen-binding domain and the V_(L) and C_(L) domains of the secondantigen-binding domain form a Fab-like antigen-binding module thatspecifically binds to the cell surface antigen, wherein the first TCRDand the second TCRD form a TCRM that is capable of recruiting at leastone TCR-associated signaling module. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the first TCRD further comprisesthe connecting peptide or a fragment thereof of the TCR β chain and/orthe second TCRD further comprises the connecting peptide or a fragmentthereof of the TCR α chain. In some embodiments, the first TCRD furthercomprises a portion of the extracellular domain of the TCR β chainand/or the second TCRD further comprises a portion of the extracellulardomain of the TCR α chain. In some embodiments, the first TCRD furthercomprises a first TCR intracellular domain and/or the second TCRDfurther comprises a second TCR intracellular domain. In someembodiments, the first TCR intracellular domain comprises a sequencefrom the intracellular domain of the TCR β chain and/or the second TCRintracellular domain comprises a sequence from the intracellular domainof the TCR α chain. In some embodiments, the abTCR further comprises atleast one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the abTCR further comprises a first signalpeptide amino-terminal to the first antigen-binding domain and/or asecond signal peptide amino-terminal to the second antigen-bindingdomain. In some embodiments, the TCRM is capable of recruiting at leastone TCR-associated signaling module selected from the group consistingof CD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotesabTCR-CD3 complex formation. In some embodiments, there is a firstpeptide linker between the first antigen-binding domain and the firstTCRD and/or a second peptide linker between the second antigen-bindingdomain and the second TCRD. In some embodiments, the first and secondpolypeptide chains are linked, such as by a covalent linkage (e.g.,peptide or other chemical linkage) or non-covalent linkage. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between a residuein the C_(H)1 antibody domain in the first antigen-binding domain and aresidue in the C_(L) antibody domain in the second antigen-bindingdomain. In some embodiments, the target antigen is a cell surfaceantigen. In some embodiments, the cell surface antigen is selected fromthe group consisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01. In some embodiments, the effector cell is a γδ T cell. Insome embodiments, the effector cell is an αβ T cell modified to block ordecrease the expression of the TCR α and/or β chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating a targetantigen-associated disease (such as cancer or viral infection) in anindividual in need thereof comprising administering to the individual aneffective amount of a composition comprising effector cells (such as Tcells or natural killer cells) presenting on their surface an abTCR thatspecifically recognizes the target antigen comprising a) a firstpolypeptide chain comprising a first antigen-binding domain comprisingV_(H) and C_(H)1 antibody domains and a first TCRD comprising thetransmembrane domain of a TCR γ chain; and b) a second polypeptide chaincomprising a second antigen-binding domain comprising V_(L) and C_(L)antibody domains and a second TCRD comprising the transmembrane domainof a TCR δ chain, wherein the V_(H) and C_(H)1 domains of the firstantigen-binding domain and the V_(L) and C_(L) domains of the secondantigen-binding domain form a Fab-like antigen-binding module thatspecifically binds to the cell surface antigen, wherein the first TCRDand the second TCRD form a TCRM that is capable of recruiting at leastone TCR-associated signaling module. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the first TCRD further comprisesthe connecting peptide or a fragment thereof of the TCR γ chain and/orthe second TCRD further comprises the connecting peptide or a fragmentthereof of the TCR δ chain. In some embodiments, the first TCRD furthercomprises a portion of the extracellular domain of the TCR γ chainand/or the second TCRD further comprises a portion of the extracellulardomain of the TCR δ chain. In some embodiments, the first TCRD furthercomprises a first TCR intracellular domain and/or the second TCRDfurther comprises a second TCR intracellular domain. In someembodiments, the first TCR intracellular domain comprises a sequencefrom the intracellular domain of the TCR γ chain and/or the second TCRintracellular domain comprises a sequence from the intracellular domainof the TCR δ chain. In some embodiments, the abTCR further comprises atleast one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the abTCR further comprises a first signalpeptide amino-terminal to the first antigen-binding domain and/or asecond signal peptide amino-terminal to the second antigen-bindingdomain. In some embodiments, the TCRM is capable of recruiting at leastone TCR-associated signaling module selected from the group consistingof CD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotesabTCR-CD3 complex formation. In some embodiments, there is a firstpeptide linker between the first antigen-binding domain and the firstTCRD and/or a second peptide linker between the second antigen-bindingdomain and the second TCRD. In some embodiments, the first and secondpolypeptide chains are linked, such as by a covalent linkage (e.g.,peptide or other chemical linkage) or non-covalent linkage. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between a residuein the C_(H)1 antibody domain in the first antigen-binding domain and aresidue in the C_(L) antibody domain in the second antigen-bindingdomain. In some embodiments, the target antigen is a cell surfaceantigen. In some embodiments, the cell surface antigen is selected fromthe group consisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01. In some embodiments, the effector cell is an αβ T cell. Insome embodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating a targetantigen-associated disease (such as cancer or viral infection) in anindividual in need thereof comprising administering to the individual aneffective amount of a composition comprising effector cells (such as Tcells or natural killer cells) presenting on their surface an abTCR thatspecifically recognizes the target antigen comprising a) a firstpolypeptide chain comprising a first antigen-binding domain comprisingV_(H) and C_(H)1 antibody domains and a first TCRD comprising thetransmembrane domain of a TCR δ chain; and b) a second polypeptide chaincomprising a second antigen-binding domain comprising V_(L) and C_(L)antibody domains and a second TCRD comprising the transmembrane domainof a TCR γ chain, wherein the V_(H) and C_(H)1 domains of the firstantigen-binding domain and the V_(L) and C_(L) domains of the secondantigen-binding domain form a Fab-like antigen-binding module thatspecifically binds to the cell surface antigen, wherein the first TCRDand the second TCRD form a TCRM that is capable of recruiting at leastone TCR-associated signaling module. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the first TCRD further comprisesthe connecting peptide or a fragment thereof of the TCR δ chain and/orthe second TCRD further comprises the connecting peptide or a fragmentthereof of the TCR γ chain. In some embodiments, the first TCRD furthercomprises a portion of the extracellular domain of the TCR δ chainand/or the second TCRD further comprises a portion of the extracellulardomain of the TCR γ chain. In some embodiments, the first TCRD furthercomprises a first TCR intracellular domain and/or the second TCRDfurther comprises a second TCR intracellular domain. In someembodiments, the first TCR intracellular domain comprises a sequencefrom the intracellular domain of the TCR δ chain and/or the second TCRintracellular domain comprises a sequence from the intracellular domainof the TCR γ chain. In some embodiments, the abTCR further comprises atleast one accessory intracellular domain comprising a T cellcostimulatory signaling sequence (such as from CD27, CD28, 4-1BB(CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA, FLAG,or myc). In some embodiments, the abTCR further comprises a first signalpeptide amino-terminal to the first antigen-binding domain and/or asecond signal peptide amino-terminal to the second antigen-bindingdomain. In some embodiments, the TCRM is capable of recruiting at leastone TCR-associated signaling module selected from the group consistingof CD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotesabTCR-CD3 complex formation. In some embodiments, there is a firstpeptide linker between the first antigen-binding domain and the firstTCRD and/or a second peptide linker between the second antigen-bindingdomain and the second TCRD. In some embodiments, the first and secondpolypeptide chains are linked, such as by a covalent linkage (e.g.,peptide or other chemical linkage) or non-covalent linkage. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between a residuein the C_(H)1 antibody domain in the first antigen-binding domain and aresidue in the C_(L) antibody domain in the second antigen-bindingdomain. In some embodiments, the target antigen is a cell surfaceantigen. In some embodiments, the cell surface antigen is selected fromthe group consisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01. In some embodiments, the effector cell is an αβ T cell. Insome embodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating a targetantigen-associated disease (such as cancer or viral infection) in anindividual in need thereof comprising administering to the individual aneffective amount of a composition comprising effector cells (such as Tcells or natural killer cells) presenting on their surface an abTCR thatspecifically recognizes the target antigen comprising a) a firstpolypeptide chain comprising, in order from amino terminus to carboxyterminus, a first antigen-binding domain and a first TCRD comprising theamino acid sequence of SEQ ID NO: 15; and b) a second polypeptide chaincomprising, in order from amino terminus to carboxy terminus, a secondantigen-binding domain and a second TCRD comprising the amino acidsequence of SEQ ID NO: 16; wherein the first antigen-binding domain andthe second antigen-binding domain form a Fab-like antigen-binding modulethat specifically binds the target antigen, wherein the first TCRD andthe second TCRD form a TCRM that is capable of recruiting at least oneTCR-associated signaling module. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the abTCR further comprises atleast one accessory intracellular domain comprising a) at least one Tcell costimulatory signaling sequence comprising (such as consisting of)the amino acid sequence of SEQ ID NO: 70 or 71; and/or b) an epitope tagcomprising (such as consisting of) the amino acid sequence of any one ofSEQ ID NOs: 50-52. In some embodiments, the abTCR further comprises afirst signal peptide amino-terminal to the first antigen-binding domainand/or a second signal peptide amino-terminal to the secondantigen-binding domain, wherein the first and/or second signal peptidescomprise the amino acid sequence of SEQ ID NO: 49. In some embodiments,the TCRM is capable of recruiting at least one TCR-associated signalingmodule selected from the group consisting of CD3δε, CD3γε, and ζζ. Insome embodiments, the TCRM promotes abTCR-CD3 complex formation. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between residuesin the C_(H)1 and C_(L) antibody domains in the Fab-like antigen-bindingmodule. In some embodiments, the target antigen is a cell surfaceantigen. In some embodiments, the cell surface antigen is selected fromthe group consisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01. In some embodiments, the effector cell is a γδ T cell. Insome embodiments, the effector cell is an αβ T cell modified to block ordecrease the expression of the TCR α and/or β chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating a targetantigen-associated disease (such as cancer or viral infection) in anindividual in need thereof comprising administering to the individual aneffective amount of a composition comprising effector cells (such as Tcells or natural killer cells) presenting on their surface an abTCR thatspecifically recognizes the target antigen comprising a) a firstpolypeptide chain comprising, in order from amino terminus to carboxyterminus, a first antigen-binding domain and a first TCRD comprising theamino acid sequence of SEQ ID NO: 17; and b) a second polypeptide chaincomprising, in order from amino terminus to carboxy terminus, a secondantigen-binding domain and a second TCRD comprising the amino acidsequence of SEQ ID NO: 18; wherein the first antigen-binding domain andthe second antigen-binding domain form a Fab-like antigen-binding modulethat specifically binds the target antigen, wherein the first TCRD andthe second TCRD form a TCRM that is capable of recruiting at least oneTCR-associated signaling module. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the abTCR further comprises atleast one accessory intracellular domain comprising a) at least one Tcell costimulatory signaling sequence comprising (such as consisting of)the amino acid sequence of SEQ ID NO: 70 or 71; and/or b) an epitope tagcomprising (such as consisting of) the amino acid sequence of any one ofSEQ ID NOs: 50-52. In some embodiments, the abTCR further comprises afirst signal peptide amino-terminal to the first antigen-binding domainand/or a second signal peptide amino-terminal to the secondantigen-binding domain, wherein the first and/or second signal peptidescomprise the amino acid sequence of SEQ ID NO: 49. In some embodiments,the TCRM is capable of recruiting at least one TCR-associated signalingmodule selected from the group consisting of CD3δε, CD3γε, and ζζ. Insome embodiments, the TCRM promotes abTCR-CD3 complex formation. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between residuesin the C_(H)1 and C_(L) antibody domains in the Fab-like antigen-bindingmodule. In some embodiments, the target antigen is a cell surfaceantigen. In some embodiments, the cell surface antigen is selected fromthe group consisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01. In some embodiments, the effector cell is a γδ T cell. Insome embodiments, the effector cell is an αβ T cell modified to block ordecrease the expression of the TCR α and/or β chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating a targetantigen-associated disease (such as cancer or viral infection) in anindividual in need thereof comprising administering to the individual aneffective amount of a composition comprising effector cells (such as Tcells or natural killer cells) presenting on their surface an abTCR thatspecifically recognizes the target antigen comprising a) a firstpolypeptide chain comprising, in order from amino terminus to carboxyterminus, a first antigen-binding domain and a first TCRD comprising theamino acid sequence of SEQ ID NO: 19; and b) a second polypeptide chaincomprising, in order from amino terminus to carboxy terminus, a secondantigen-binding domain and a second TCRD comprising the amino acidsequence of SEQ ID NO: 20; wherein the first antigen-binding domain andthe second antigen-binding domain form a Fab-like antigen-binding modulethat specifically binds the target antigen, wherein the first TCRD andthe second TCRD form a TCRM that is capable of recruiting at least oneTCR-associated signaling module. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the abTCR further comprises atleast one accessory intracellular domain comprising a) at least one Tcell costimulatory signaling sequence comprising (such as consisting of)the amino acid sequence of SEQ ID NO: 70 or 71; and/or b) an epitope tagcomprising (such as consisting of) the amino acid sequence of any one ofSEQ ID NOs: 50-52. In some embodiments, the abTCR further comprises afirst signal peptide amino-terminal to the first antigen-binding domainand/or a second signal peptide amino-terminal to the secondantigen-binding domain, wherein the first and/or second signal peptidescomprise the amino acid sequence of SEQ ID NO: 49.

In some embodiments, the TCRM is capable of recruiting at least oneTCR-associated signaling module selected from the group consisting ofCD3δε, CD3γε, and ζζ. In some embodiments, the TCRM promotes abTCR-CD3complex formation. In some embodiments, the first polypeptide chain andthe second polypeptide chain are linked via a) a disulfide bond betweena residue in the connecting peptide of the first TCRD and a residue inthe connecting peptide of the second TCRD; and/or b) a disulfide bondbetween residues in the C_(H)1 and C_(L) antibody domains in theFab-like antigen-binding module. In some embodiments, the target antigenis a cell surface antigen. In some embodiments, the cell surface antigenis selected from the group consisting of a protein, a carbohydrate, anda lipid. In some embodiments, the cell surface antigen is adisease-associated antigen, such as a tumor-associated orvirally-encoded antigen. In some embodiments, the cell surface antigenis CD19, ROR1, ROR2, BCMA, GPRC5D, or FCRL5. In some embodiments, thetarget antigen is a surface-presented peptide/MHC complex. In someembodiments, the peptide/MHC complex comprises a peptide derived from adisease-associated antigen (such as a tumor-associated orvirally-encoded antigen) and an MHC protein. In some embodiments, thepeptide/MHC complex comprises a peptide and an MHC protein, wherein thepeptide is derived from a protein selected from the group consisting ofWT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, and PSA. In someembodiments, the MHC protein is an MHC class I protein. In someembodiments, the MHC class I protein is HLA-A. In some embodiments, theHLA-A is HLA-A02. In some embodiments, the HLA-A02 is HLA-A*02:01. Insome embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating a targetantigen-associated disease (such as cancer or viral infection) in anindividual in need thereof comprising administering to the individual aneffective amount of a composition comprising effector cells (such as Tcells or natural killer cells) presenting on their surface an abTCR thatspecifically recognizes the target antigen comprising a) a firstpolypeptide chain comprising, in order from amino terminus to carboxyterminus, a first antigen-binding domain and a first TCRD comprising theamino acid sequence of SEQ ID NO: 21; and b) a second polypeptide chaincomprising, in order from amino terminus to carboxy terminus, a secondantigen-binding domain and a second TCRD comprising the amino acidsequence of SEQ ID NO: 22; wherein the first antigen-binding domain andthe second antigen-binding domain form a Fab-like antigen-binding modulethat specifically binds the target antigen, wherein the first TCRD andthe second TCRD form a TCRM that is capable of recruiting at least oneTCR-associated signaling module. In some embodiments, the Fab-likeantigen-binding module is human, humanized, chimeric, semi-synthetic, orfully synthetic. In some embodiments, the abTCR further comprises atleast one accessory intracellular domain comprising a) at least one Tcell costimulatory signaling sequence comprising (such as consisting of)the amino acid sequence of SEQ ID NO: 70 or 71; and/or b) an epitope tagcomprising (such as consisting of) the amino acid sequence of any one ofSEQ ID NOs: 50-52. In some embodiments, the abTCR further comprises afirst signal peptide amino-terminal to the first antigen-binding domainand/or a second signal peptide amino-terminal to the secondantigen-binding domain, wherein the first and/or second signal peptidescomprise the amino acid sequence of SEQ ID NO: 49. In some embodiments,the TCRM is capable of recruiting at least one TCR-associated signalingmodule selected from the group consisting of CD3δε, CD3γε, and ζζ. Insome embodiments, the TCRM promotes abTCR-CD3 complex formation. In someembodiments, the first polypeptide chain and the second polypeptidechain are linked via a) a disulfide bond between a residue in theconnecting peptide of the first TCRD and a residue in the connectingpeptide of the second TCRD; and/or b) a disulfide bond between residuesin the C_(H)1 and C_(L) antibody domains in the Fab-like antigen-bindingmodule. In some embodiments, the target antigen is a cell surfaceantigen. In some embodiments, the cell surface antigen is selected fromthe group consisting of a protein, a carbohydrate, and a lipid. In someembodiments, the cell surface antigen is a disease-associated antigen,such as a tumor-associated or virally-encoded antigen. In someembodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D,or FCRL5. In some embodiments, the target antigen is a surface-presentedpeptide/MHC complex. In some embodiments, the peptide/MHC complexcomprises a peptide derived from a disease-associated antigen (such as atumor-associated or virally-encoded antigen) and an MHC protein. In someembodiments, the peptide/MHC complex comprises a peptide and an MHCprotein, wherein the peptide is derived from a protein selected from thegroup consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A,HIV-1, and PSA. In some embodiments, the MHC protein is an MHC class Iprotein. In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 isHLA-A*02:01. In some embodiments, the effector cell is an αβ T cell. Insome embodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating anAFP-associated disease in an individual in need thereof comprisingadministering to the individual an effective amount of a compositioncomprising effector cells (such as T cells or natural killer cells)presenting on their surface an abTCR comprising a) a first polypeptidechain comprising a first abTCR domain comprising the amino acid sequenceof SEQ ID NO: 23; and b) a second polypeptide chain comprising a secondabTCR domain comprising the amino acid sequence of SEQ ID NO: 24,wherein the first polypeptide chain and the second polypeptide chain arelinked via one or more disulfide bonds. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga T cell costimulatory signaling sequence (such as from CD27, CD28,4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA,FLAG, or myc). In some embodiments, the epitope tag comprises any one ofthe amino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is a γδ T cell. In someembodiments, the effector cell is an αβ T cell modified to block ordecrease the expression of the TCR α and/or β chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating anAFP-associated disease in an individual in need thereof comprisingadministering to the individual an effective amount of a compositioncomprising effector cells (such as T cells or natural killer cells)presenting on their surface an abTCR comprising a) a first polypeptidechain comprising a first abTCR domain comprising the amino acid sequenceof SEQ ID NO: 25; and b) a second polypeptide chain comprising a secondabTCR domain comprising the amino acid sequence of SEQ ID NO: 26,wherein the first polypeptide chain and the second polypeptide chain arelinked via one or more disulfide bonds. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga T cell costimulatory signaling sequence (such as from CD27, CD28,4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA,FLAG, or myc). In some embodiments, the epitope tag comprises any one ofthe amino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is a γδ T cell. In someembodiments, the effector cell is an αβ T cell modified to block ordecrease the expression of the TCR α and/or β chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating anAFP-associated disease in an individual in need thereof comprisingadministering to the individual an effective amount of a compositioncomprising effector cells (such as T cells or natural killer cells)presenting on their surface an abTCR comprising a) a first polypeptidechain comprising a first abTCR domain comprising the amino acid sequenceof SEQ ID NO: 27; and b) a second polypeptide chain comprising a secondabTCR domain comprising the amino acid sequence of SEQ ID NO: 28,wherein the first polypeptide chain and the second polypeptide chain arelinked via one or more disulfide bonds. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga T cell costimulatory signaling sequence (such as from CD27, CD28,4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA,FLAG, or myc). In some embodiments, the epitope tag comprises any one ofthe amino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is a γδ T cell. In someembodiments, the effector cell is an αβ T cell modified to block ordecrease the expression of the TCR α and/or β chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating anAFP-associated disease in an individual in need thereof comprisingadministering to the individual an effective amount of a compositioncomprising effector cells (such as T cells or natural killer cells)presenting on their surface an abTCR comprising a) a first polypeptidechain comprising a first abTCR domain comprising the amino acid sequenceof SEQ ID NO: 29; and b) a second polypeptide chain comprising a secondabTCR domain comprising the amino acid sequence of SEQ ID NO: 30,wherein the first polypeptide chain and the second polypeptide chain arelinked via one or more disulfide bonds. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga T cell costimulatory signaling sequence (such as from CD27, CD28,4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA,FLAG, or myc). In some embodiments, the epitope tag comprises any one ofthe amino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating anAFP-associated disease in an individual in need thereof comprisingadministering to the individual an effective amount of a compositioncomprising effector cells (such as T cells or natural killer cells)presenting on their surface an abTCR comprising a) a first polypeptidechain comprising a first abTCR domain comprising the amino acid sequenceof SEQ ID NO: 31; and b) a second polypeptide chain comprising a secondabTCR domain comprising the amino acid sequence of SEQ ID NO: 32,wherein the first polypeptide chain and the second polypeptide chain arelinked via one or more disulfide bonds. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga T cell costimulatory signaling sequence (such as from CD27, CD28,4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA,FLAG, or myc). In some embodiments, the epitope tag comprises any one ofthe amino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating anAFP-associated disease in an individual in need thereof comprisingadministering to the individual an effective amount of a compositioncomprising effector cells (such as T cells or natural killer cells)presenting on their surface an abTCR comprising a) a first polypeptidechain comprising a first abTCR domain comprising the amino acid sequenceof SEQ ID NO: 33; and b) a second polypeptide chain comprising a secondabTCR domain comprising the amino acid sequence of SEQ ID NO: 34,wherein the first polypeptide chain and the second polypeptide chain arelinked via one or more disulfide bonds. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga T cell costimulatory signaling sequence (such as from CD27, CD28,4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA,FLAG, or myc). In some embodiments, the epitope tag comprises any one ofthe amino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating anAFP-associated disease in an individual in need thereof comprisingadministering to the individual an effective amount of a compositioncomprising effector cells (such as T cells or natural killer cells)presenting on their surface an abTCR comprising a) a first polypeptidechain comprising a first abTCR domain comprising the amino acid sequenceof SEQ ID NO: 35; and b) a second polypeptide chain comprising a secondabTCR domain comprising the amino acid sequence of SEQ ID NO: 36,wherein the first polypeptide chain and the second polypeptide chain arelinked via one or more disulfide bonds. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga T cell costimulatory signaling sequence (such as from CD27, CD28,4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA,FLAG, or myc). In some embodiments, the epitope tag comprises any one ofthe amino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating aCD19-associated disease in an individual in need thereof comprisingadministering to the individual an effective amount of a compositioncomprising effector cells (such as T cells or natural killer cells)presenting on their surface an abTCR comprising a) a first polypeptidechain comprising a first abTCR domain comprising the amino acid sequenceof SEQ ID NO: 42; and b) a second polypeptide chain comprising a secondabTCR domain comprising the amino acid sequence of SEQ ID NO: 43,wherein the first polypeptide chain and the second polypeptide chain arelinked via one or more disulfide bonds. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga T cell costimulatory signaling sequence (such as from CD27, CD28,4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA,FLAG, or myc). In some embodiments, the epitope tag comprises any one ofthe amino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating aCD19-associated disease in an individual in need thereof comprisingadministering to the individual an effective amount of a compositioncomprising effector cells (such as T cells or natural killer cells)presenting on their surface an abTCR comprising a) a first polypeptidechain comprising a first abTCR domain comprising the amino acid sequenceof SEQ ID NO: 42; and b) a second polypeptide chain comprising a secondabTCR domain comprising the amino acid sequence of SEQ ID NO: 54,wherein the first polypeptide chain and the second polypeptide chain arelinked via one or more disulfide bonds. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga T cell costimulatory signaling sequence (such as from CD27, CD28,4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA,FLAG, or myc). In some embodiments, the epitope tag comprises any one ofthe amino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating aCD19-associated disease in an individual in need thereof comprisingadministering to the individual an effective amount of a compositioncomprising effector cells (such as T cells or natural killer cells)presenting on their surface an abTCR comprising a) a first polypeptidechain comprising a first abTCR domain comprising the amino acid sequenceof SEQ ID NO: 55; and b) a second polypeptide chain comprising a secondabTCR domain comprising the amino acid sequence of SEQ ID NO: 54,wherein the first polypeptide chain and the second polypeptide chain arelinked via one or more disulfide bonds. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga T cell costimulatory signaling sequence (such as from CD27, CD28,4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA,FLAG, or myc). In some embodiments, the epitope tag comprises any one ofthe amino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

In some embodiments, there is provided a method of treating aCD19-associated disease in an individual in need thereof comprisingadministering to the individual an effective amount of a compositioncomprising effector cells (such as T cells or natural killer cells)presenting on their surface an abTCR comprising a) a first polypeptidechain comprising a first abTCR domain comprising the amino acid sequenceof SEQ ID NO: 56; and b) a second polypeptide chain comprising a secondabTCR domain comprising the amino acid sequence of SEQ ID NO: 54,wherein the first polypeptide chain and the second polypeptide chain arelinked via one or more disulfide bonds. In some embodiments, the abTCRfurther comprises at least one accessory intracellular domain comprisinga T cell costimulatory signaling sequence (such as from CD27, CD28,4-1BB (CD137), OX40, CD30, or CD40) and/or an epitope tag (such as HA,FLAG, or myc). In some embodiments, the epitope tag comprises any one ofthe amino acid sequences of SEQ ID NOs: 50-52. In some embodiments, thefirst polypeptide chain further comprises a first signal peptide aminoterminal to the first abTCR domain and/or the second polypeptide chainfurther comprises a second signal peptide amino terminal to the secondabTCR domain. In some embodiments, the first and/or second signalpeptides comprise (such as consist of) the amino acid sequence of SEQ IDNO: 49. In some embodiments, the effector cell is an αβ T cell. In someembodiments, the effector cell is a γδ T cell modified to block ordecrease the expression of the TCR γ and/or δ chains. In someembodiments, the effector cell is selected from the group consisting ofa cytotoxic T cell, a helper T cell, a natural killer T cell, and asuppressor T cell.

Also contemplated are methods of treating a target antigen-associateddisease in an individual in need thereof comprising administering to theindividual a composition comprising a plurality of effector cellsexpressing different abTCRs. Thus, in some embodiments, according to anyof the methods for treating a target antigen-associated disease in anindividual described herein, the composition is a heterogeneous abTCReffector cell composition as described herein.

For example, in some embodiments, there is provided a method of treatinga target antigen-associated disease (such as cancer or viral infection)in an individual in need thereof comprising administering to theindividual an effective amount of a heterogeneous abTCR effector cellcomposition comprising a plurality of abTCR effector cell populationsaccording to any of the embodiments described herein, wherein all of theabTCR effector cells in the composition are of the same cell type (e.g.,all of the abTCR effector cells are cytotoxic T cells), wherein eachpopulation of abTCR effector cells expresses a different abTCR than theothers, and wherein at least one population of abTCR effector cellsexpresses an abTCR that specifically binds to the target antigen. Insome embodiments, the abTCR effector cells are T cells. In someembodiments, the abTCR effector cells are selected from the groupconsisting of cytotoxic T cells, helper T cells, natural killer T cells,and suppressor T cells. In some embodiments, each population of abTCReffector cells expresses an abTCR that specifically binds to the targetantigen. In some embodiments, at least one population of abTCR effectorcells expresses an abTCR that specifically binds to a different targetantigen. In some embodiments, where at least one population of abTCReffector cells expresses an abTCR that specifically binds to a differenttarget antigen, each of the different target antigens is associated withthe target antigen-associated disease.

In some embodiments, there is provided a method of treating a targetantigen-associated disease (such as cancer or viral infection) in anindividual in need thereof comprising administering to the individual aneffective amount of a heterogeneous abTCR effector cell compositioncomprising a plurality of abTCR effector cell populations according toany of the embodiments described herein, wherein at least one populationof abTCR effector cells is of a different cell type than the others, andwherein at least one population of abTCR effector cells expresses anabTCR that specifically binds to the target antigen. In someembodiments, all of the populations of abTCR effector cells are ofdifferent cell types. In some embodiments, the abTCR effector cells areT cells. In some embodiments, each population of abTCR effector cells isof a cell type selected from the group consisting of cytotoxic T cells,helper T cells, natural killer T cells, and suppressor T cells. In someembodiments, each population of abTCR effector cells expresses the sameabTCR. In some embodiments, at least one population of abTCR effectorcells expresses a different abTCR than the others. In some embodiments,each population of abTCR effector cells expresses a different abTCR thanthe others. In some embodiments, each population of abTCR effector cellsexpresses an abTCR that specifically binds to the target antigen. Insome embodiments, at least one population of abTCR effector cellsexpresses an abTCR that specifically binds to a different targetantigen. In some embodiments, where at least one population of abTCReffector cells expresses an abTCR that specifically binds to a differenttarget antigen, each of the different target antigens is associated withthe target antigen-associated disease.

In some embodiments, there is provided a method of treating a diseaseassociated with a plurality of target antigens in an individual in needthereof comprising administering to the individual an effective amountof a heterogeneous abTCR effector cell composition comprising aplurality of abTCR effector cell populations according to any of theembodiments described herein, wherein all of the abTCR effector cells inthe composition are of the same cell type (e.g., all of the abTCReffector cells are cytotoxic T cells), wherein each population of abTCReffector cells expresses a different abTCR than the others, and whereinfor each target antigen of the plurality of target antigens, at leastone population of abTCR effector cells expresses an abTCR thatspecifically binds to the target antigen. In some embodiments, the abTCReffector cells are T cells. In some embodiments, the abTCR effectorcells are selected from the group consisting of cytotoxic T cells,helper T cells, natural killer T cells, and suppressor T cells.

In some embodiments, there is provided a method of treating a diseaseassociate with a plurality of target antigens in an individual in needthereof comprising administering to the individual an effective amountof a heterogeneous abTCR effector cell composition comprising aplurality of abTCR effector cell populations according to any of theembodiments described herein, wherein at least one population of abTCReffector cells is of a different cell type than the others, and whereinfor each target antigen of the plurality of target antigens, at leastone population of abTCR effector cells expresses an abTCR thatspecifically binds to the target antigen. In some embodiments, all ofthe populations of abTCR effector cells are of different cell types. Insome embodiments, the abTCR effector cells are T cells. In someembodiments, each population of abTCR effector cells is of a cell typeselected from the group consisting of cytotoxic T cells, helper T cells,natural killer T cells, and suppressor T cells. In some embodiments,each population of abTCR effector cells expresses a different abTCR thanthe others.

In some embodiments, the individual is a mammal (e.g., human, non-humanprimate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). Insome embodiments, the individual is a human. In some embodiments, theindividual is a clinical patient, a clinical trial volunteer, anexperimental animal, etc. In some embodiments, the individual is youngerthan about 60 years old (including for example younger than about any of50, 40, 30, 25, 20, 15, or 10 years old). In some embodiments, theindividual is older than about 60 years old (including for example olderthan about any of 70, 80, 90, or 100 years old). In some embodiments,the individual is diagnosed with or environmentally or genetically proneto one or more of the diseases or disorders described herein (such ascancer or viral infection). In some embodiments, the individual has oneor more risk factors associated with one or more diseases or disordersdescribed herein.

In some embodiments, the abTCR effector cell compositions of theinvention are administered in combination with a second, third, orfourth agent (including, e.g., an antineoplastic agent, a growthinhibitory agent, a cytotoxic agent, or a chemotherapeutic agent) totreat diseases or disorders involving target antigen expression. In someembodiments, the abTCR effector cell composition is administered incombination with a cytokine (such as IL-2). In some embodiments, theabTCR is administered in combination with an agent that increases theexpression of MHC proteins and/or enhances the surface presentation ofpeptides by MHC proteins. In some embodiments, the agent includes, forexample, IFN receptor agonists, Hsp90 inhibitors, enhancers of p53expression, and chemotherapeutic agents. In some embodiments, the agentis an IFN receptor agonist including, for example, IFNγ, IFNβ, and IFNα.In some embodiments, the agent is an Hsp90 inhibitor including, forexample, tanespimycin (17-AAG), alvespimycin (17-DMAG), retaspimycin(IPI-504), IPI-493, CNF2024/BIIB021, MPC-3100, Debio 0932 (CUDC-305),PU-H71, Ganetespib (STA-9090), NVP-AUY922 (VER-52269), HSP990, KW-2478,AT13387, SNX-5422, DS-2248, and XL888. In some embodiments, the agent isan enhancer of p53 expression including, for example, 5-fluorouracil andnutlin-3. In some embodiments, the agent is a chemotherapeutic agentincluding, for example, topotecan, etoposide, cisplatin, paclitaxel, andvinblastine.

In some embodiments, there is provided a method of treating a targetantigen-positive disease in an individual in need thereof comprisingadministering to the individual an abTCR effector cell compositionaccording to any of the embodiments described herein in combination witha cytokine (such as IL-2). In some embodiments, the abTCR effector cellcomposition and the cytokine are administered simultaneously. In someembodiments, the abTCR effector cell composition and the cytokine areadministered sequentially.

In some embodiments, there is provided a method of treating a targetantigen-positive disease in an individual in need thereof, wherein thecells expressing the target antigen do not normally present, or presentat relatively low levels, a complex comprising the target antigen and anMHC class I protein on their surface, the method comprisingadministering to the individual an abTCR effector cell compositionsaccording to any of the embodiments described herein in combination withan agent that increases the expression of MHC class I proteins and/orenhances the surface presentation of target antigens by MHC class Iproteins. In some embodiments, the agent includes, for example, IFNreceptor agonists, Hsp90 inhibitors, enhancers of p53 expression, andchemotherapeutic agents. In some embodiments, the agent is an IFNreceptor agonist including, for example, IFNγ, IFNβ, and IFNα. In someembodiments, the agent is an Hsp90 inhibitor including, for example,tanespimycin (17-AAG), alvespimycin (17-DMAG), retaspimycin (IPI-504),IPI-493, CNF2024/BIIB021, MPC-3100, Debio 0932 (CUDC-305), PU-H71,Ganetespib (STA-9090), NVP-AUY922 (VER-52269), HSP990, KW-2478, AT13387,SNX-5422, DS-2248, and XL888. In some embodiments, the agent is anenhancer of p53 expression including, for example, 5-fluorouracil andnutlin-3. In some embodiments, the agent is a chemotherapeutic agentincluding, for example, topotecan, etoposide, cisplatin, paclitaxel, andvinblastine. In some embodiments, the abTCR effector cell compositionand the agent are administered simultaneously. In some embodiments, theabTCR effector cell composition and the agent are administeredsequentially.

In some embodiments, there is provided a method of treating a targetantigen-associated disease (such as cancer or viral infection) in anindividual in need thereof comprising administering to the individual aneffective amount of a composition comprising nucleic acid encoding anabTCR according to any of the embodiments described herein. Methods forgene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346,5,580,859, 5,589,466, incorporated by reference herein in theirentireties.

Cancer treatments can be evaluated, for example, by tumor regression,tumor weight or size shrinkage, time to progression, duration ofsurvival, progression free survival, overall response rate, duration ofresponse, quality of life, protein expression and/or activity.Approaches to determining efficacy of the therapy can be employed,including for example, measurement of response through radiologicalimaging.

In some embodiments, the efficacy of treatment is measured as thepercentage tumor growth inhibition (% TGI), calculated using theequation 100-(T/C×100), where T is the mean relative tumor volume of thetreated tumor, and C is the mean relative tumor volume of a non-treatedtumor. In some embodiments, the % TGI is about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, or more than 95%.

Viral infection treatments can be evaluated, for example, by viral load,duration of survival, quality of life, protein expression and/oractivity.

Diseases

The abTCR effector cells in some embodiments can be useful for treatingcancers associated with a target antigen. Cancers that may be treatedusing any of the methods described herein include tumors that are notvascularized, or not yet substantially vascularized, as well asvascularized tumors. The cancers may comprise non-solid tumors (such ashematological tumors, for example, leukemias and lymphomas) or maycomprise solid tumors. Types of cancers to be treated with the abTCReffector cells of the invention include, but are not limited to,carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoidmalignancies, benign and malignant tumors, and malignancies e.g.,sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatrictumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, plasmacytoma, Waldenstrom'smacroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairycell leukemia and myelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include adrenocorticalcarcinoma, cholangiocarcinoma, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteosarcoma, and other sarcomas, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, stomach cancer, lymphoid malignancy, pancreatic cancer,breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, thyroid cancer (e.g., medullarythyroid carcinoma and papillary thyroid carcinoma), pheochromocytomassebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cellcarcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor,cervical cancer (e.g., cervical carcinoma and pre-invasive cervicaldysplasia), colorectal cancer, cancer of the anus, anal canal, oranorectum, vaginal cancer, cancer of the vulva (e.g., squamous cellcarcinoma, intraepithelial carcinoma, adenocarcinoma, and fibrosarcoma),penile cancer, oropharyngeal cancer, esophageal cancer, head cancers(e.g., squamous cell carcinoma), neck cancers (e.g., squamous cellcarcinoma), testicular cancer (e.g., seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, Leydig cell tumor,fibroma, fibroadenoma, adenomatoid tumors, and lipoma), bladdercarcinoma, kidney cancer, melanoma, cancer of the uterus (e.g.,endometrial carcinoma), urothelial cancers (e.g., squamous cellcarcinoma, transitional cell carcinoma, adenocarcinoma, ureter cancer,and urinary bladder cancer), and CNS tumors (such as a glioma (such asbrainstem glioma and mixed gliomas), glioblastoma (also known asglioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma,medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,neuroblastoma, retinoblastoma and brain metastases).

Cancer treatments can be evaluated, for example, by tumor regression,tumor weight or size shrinkage, time to progression, duration ofsurvival, progression free survival, overall response rate, duration ofresponse, quality of life, protein expression and/or activity.Approaches to determining efficacy of the therapy can be employed,including for example, measurement of response through radiologicalimaging.

The abTCR effector cells in other embodiments can be useful for treatinginfectious diseases by targeting pathogen-associated (such asvirally-encoded) antigens. The infection to be prevented or treated, forexample, may be caused by a virus, bacteria, protozoa, or parasite. Thetarget antigen may be a pathogenic protein, polypeptide or peptide thatis responsible for a disease caused by the pathogen, or is capable ofinducing an immunological response in a host infected by the pathogen.Pathogenic antigens which can be targeted by abTCR effector cellsinclude, but are not limited to, antigens derived from Acinetobacterbaumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostomabraziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum,Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus,Bacillus anthracia, Bacillus cereus, Bartonella henselae, BK virus,Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis,Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus,Brugia malayi, Bunyaviridae family, Burkholderia cepacia and otherBurkholderia species, Burkholderia mallei, Burkholderia pseudomallei,Caliciviridae family, Campylobacter genus, Candida albicans, Candidaspp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophilapsittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum,Clostridium difficile, Clostridium perfringens, Clostridium perfringens,Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses,Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congohemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus,Cytomegalovirus (CMV), Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4),Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichiachaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica,Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie Avirus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus(EBV), Escherichia coli O157:H7, 0111 and 0104:H4, Fasciola hepatica andFasciola gigantica, FFI prion, Filarioidea superfamily, Flaviviruses,Francisella tularensis, Fusobacterium genus, Geotrichum candidum,Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus,Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori,Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis BVirus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis EVirus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasmacapsulatum, HIV (Human immunodeficiency virus), Hortaea werneckii, Humanbocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7(HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Humanparainfluenza viruses (HPIV), Human T cell leukemia virus 1 (HTLV-1),Japanese encephalitis virus, JC virus, Junin virus, Kaposi's Sarcomaassociated herpesvirus (KSHV), Kingella kingae, Klebsiella granulomatis,Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus,Leptospira genus, Listeria monocytogenes, Lymphocytic choriomeningitisvirus (LCMV), Machupo virus, Malassezia spp, Marburg virus, Measlesvirus, Metagonimus yokagawai, Microsporidia phylum, Molluscumcontagiosum virus (MCV), Mumps virus, Mycobacterium leprae andMycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacteriumulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus,Neisseria gonorrhoeae, Neisseria meningitidis, Nocardiai asteroides,Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi,Orthomyxoviridae family (Influenza), Paracoccidioides brasiliensis,Paragonimus spp, Paragonimus westermani, Parvovirus B19, Pasteurellagenus, Plasmodium genus, Pneumocystis jirovecii, Poliovirus, Rabiesvirus, Respiratory syncytial virus (RSV), Rhinovirus, rhinoviruses,Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsiarickettsii, Rickettsia typhi, Rift Valley fever virus, Rotavirus,Rubella virus, Sabia virus, Salmonella genus, Sarcoptes scabiei, SARScoronavirus, Schistosoma genus, Shigella genus, Sin Nombre virus,Hantavirus, Sporothrix schenckii, Staphylococcus genus, Staphylococcusgenus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcuspyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium,Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati,Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonasvaginalis, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei,Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV),Varicella zoster virus (VZV), Variola major or Variola minor, vCJDprion, Venezuelan equine encephalitis virus, Vibrio cholerae, West Nilevirus, Western equine encephalitis virus, Wuchereria bancrofti, Yellowfever virus, Yersinia enterocolitica, Yersinia pestis, and Yersiniapseudotuberculosis.

In some embodiments, the abTCR effector cells are used for treatingoncogenic infectious diseases, such as infection by oncogenic viruses.Oncogenic viruses include, but are not limited to, CMV, EBV, HBV, KSHV,HPV, MCV, HTLV-1, HIV-1, and HCV. The target antigen of the abTCR can bea viral oncoprotein including, but not limited to, Tax, E7, E6/E7, E6,HBx, EBNA proteins (e.g., EBNA3 A, EBNA3 C, and EBNA 2), v-cyclin,LANA1, LANA2, LMP-1, k-bZIP, RTA, KSHV K8, and fragments thereof. SeeAhuja, Richa, et al., Curr. Sci., 2014.

Articles of Manufacture and Kits

In some embodiments of the invention, there is provided an article ofmanufacture containing materials useful for the treatment of a targetantigen-positive disease such as cancer (for example adrenocorticalcarcinoma, bladder cancer, breast cancer, cervical cancer,cholangiocarcinoma, colorectal cancers, esophageal cancer, glioblastoma,glioma, hepatocellular carcinoma, head and neck cancer, kidney cancer,lung cancer, melanoma, mesothelioma, multiple myeloma, pancreaticcancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer,prostate cancer, sarcoma, stomach cancer, uterine cancer or thyroidcancer) or viral infection (for example infection by CMV, EBV, HBV,KSHV, HPV, MCV, HTLV-1, HIV-1, or HCV). The article of manufacture cancomprise a container and a label or package insert on or associated withthe container. Suitable containers include, for example, bottles, vials,syringes, etc. The containers may be formed from a variety of materialssuch as glass or plastic. Generally, the container holds a compositionwhich is effective for treating a disease or disorder described herein,and may have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). At least one active agent in thecomposition is an effector cell presenting on its surface an abTCR ofthe invention. The label or package insert indicates that thecomposition is used for treating the particular condition. The label orpackage insert will further comprise instructions for administering theabTCR effector cell composition to the patient. Articles of manufactureand kits comprising combinatorial therapies described herein are alsocontemplated.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products. In someembodiments, the package insert indicates that the composition is usedfor treating a target antigen-positive cancer (such as adrenocorticalcarcinoma, bladder cancer, breast cancer, cervical cancer,cholangiocarcinoma, colorectal cancers, esophageal cancer, glioblastoma,glioma, hepatocellular carcinoma, head and neck cancer, kidney cancer,lung cancer, melanoma, mesothelioma, multiple myeloma, pancreaticcancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer,prostate cancer, sarcoma, stomach cancer, uterine cancer or thyroidcancer). In other embodiments, the package insert indicates that thecomposition is used for treating a target antigen-positive viralinfection (for example infection by CMV, EBV, HBV, KSHV, HPV, MCV,HTLV-1, HIV-1, or HCV).

Additionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, e.g., fortreatment of a target antigen-positive disease or disorder describedherein, optionally in combination with the articles of manufacture. Kitsof the invention include one or more containers comprising an abTCReffector cell composition (or unit dosage form and/or article ofmanufacture), and in some embodiments, further comprise another agent(such as the agents described herein) and/or instructions for use inaccordance with any of the methods described herein. The kit may furthercomprise a description of selection of individuals suitable fortreatment. Instructions supplied in the kits of the invention aretypically written instructions on a label or package insert (e.g., apaper sheet included in the kit), but machine-readable instructions(e.g., instructions carried on a magnetic or optical storage disk) arealso acceptable.

For example, in some embodiments, the kit comprises a compositioncomprising an effector cell presenting on its surface an abTCR. In someembodiments, the kit comprises a) a composition comprising an effectorcell presenting on its surface an abTCR, and b) an effective amount ofat least one other agent, wherein the other agent increases theexpression of MHC proteins and/or enhances the surface presentation ofpeptides by MHC proteins (e.g., IFNγ, IFNβ, IFNα, or Hsp90 inhibitor).In some embodiments, the kit comprises a) a composition comprising aneffector cell presenting on its surface an abTCR, and b) instructionsfor administering the abTCR effector cell composition to an individualfor treatment of a target antigen-positive disease (such as cancer orviral infection). In some embodiments, the kit comprises a) acomposition comprising an effector cell presenting on its surface anabTCR, b) an effective amount of at least one other agent, wherein theother agent increases the expression of MHC proteins and/or enhances thesurface presentation of peptides by MHC proteins (e.g., IFNγ, IFNβ,IFNα, or Hsp90 inhibitor), and c) instructions for administering theabTCR effector cell composition and the other agent(s) to an individualfor treatment of a target antigen-positive disease (such as cancer orviral infection). The abTCR effector cell composition and the otheragent(s) can be present in separate containers or in a single container.For example, the kit may comprise one distinct composition or two ormore compositions wherein one composition comprises the abTCR effectorcell and another composition comprises the other agent.

In some embodiments, the kit comprises a) a composition comprising anabTCR, and b) instructions for combining the abTCR with effector cells(such as effector cells, e.g., T cells or natural killer cells, derivedfrom an individual) to form a composition comprising the effector cellspresenting on their surface the abTCR and administering the abTCReffector cell composition to the individual for treatment of a targetantigen-positive disease (such as cancer or viral infection). In someembodiments, the kit comprises a) a composition comprising an abTCR, andb) an effector cell (such as a cytotoxic cell). In some embodiments, thekit comprises a) a composition comprising an abTCR, b) an effector cell(such as a cytotoxic cell), and c) instructions for combining the abTCRwith the effector cell to form a composition comprising the effectorcell presenting on its surface the abTCR and administering the abTCReffector cell composition to an individual for the treatment of a targetantigen-positive disease (such as cancer or viral infection).

In some embodiments, the kit comprises a nucleic acid (or set of nucleicacids) encoding an abTCR. In some embodiments, the kit comprises a) anucleic acid (or set of nucleic acids) encoding an abTCR, and b) a hostcell (such as an effector cell) for expressing the nucleic acid (or setof nucleic acids). In some embodiments, the kit comprises a) a nucleicacid (or set of nucleic acids) encoding an abTCR, and b) instructionsfor i) expressing the abTCR in a host cell (such as an effector cell,e.g., a T cell), ii) preparing a composition comprising the host cellexpressing the abTCR, and iii) administering the composition comprisingthe host cell expressing the abTCR to an individual for the treatment ofa target antigen-positive disease (such as cancer or viral infection).In some embodiments, the host cell is derived from the individual. Insome embodiments, the kit comprises a) a nucleic acid (or set of nucleicacids) encoding an abTCR, b) a host cell (such as an effector cell) forexpressing the nucleic acid (or set of nucleic acids), and c)instructions for i) expressing the abTCR in the host cell, ii) preparinga composition comprising the host cell expressing the abTCR, and iii)administering the composition comprising the host cell expressing theabTCR to an individual for the treatment of a target antigen-positivedisease (such as cancer or viral infection).

The kits of the invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., seled Mylar or plastic bags), and the like. Kits mayoptionally provide additional components such as buffers andinterpretative information. The present application thus also providesarticles of manufacture, which include vials (such as sealed vials),bottles, jars, flexible packaging, and the like.

The instructions relating to the use of the abTCR effector cellcompositions generally include information as to dosage, dosingschedule, and route of administration for the intended treatment. Thecontainers may be unit doses, bulk packages (e.g., multi-dose packages)or sub-unit doses. For example, kits may be provided that containsufficient dosages of an abTCR effector cell composition as disclosedherein to provide effective treatment of an individual for an extendedperiod, such as any of a week, 8 days, 9 days, 10 days, 11 days, 12days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4months, 5 months, 7 months, 8 months, 9 months, or more. Kits may alsoinclude multiple unit doses of the abTCR and pharmaceutical compositionsand instructions for use and packaged in quantities sufficient forstorage and use in pharmacies, for example, hospital pharmacies andcompounding pharmacies.

Those skilled in the art will recognize that several embodiments arepossible within the scope and spirit of this invention. The inventionwill now be described in greater detail by reference to the followingnon-limiting examples. The following examples further illustrate theinvention but, of course, should not be construed as in any way limitingits scope.

EXEMPLARY EMBODIMENTS Embodiment 1

An antibody-T cell receptor (TCR) chimeric molecule (abTCR) thatspecifically binds to a target antigen, comprising:

a) a first polypeptide chain comprising a first antigen-binding domaincomprising V_(H) and C_(H)1 antibody domains and a first T cell receptordomain (TCRD) comprising a first transmembrane domain of a first TCRsubunit; and

b) a second polypeptide chain comprising a second antigen-binding domaincomprising V_(L) and C_(L) antibody domains and a second TCRD comprisinga second transmembrane domain of a second TCR subunit,

wherein the V_(H) and C_(H)1 domains of the first antigen-binding domainand the V_(L) and C_(L) domains of the second antigen-binding domainform an antigen-binding module that specifically binds to the targetantigen,

and wherein the first TCRD and the second TCRD form a T cell receptormodule (TCRM) that is capable of recruiting at least one TCR-associatedsignaling module.

Embodiment 2

The abTCR of embodiment 1, wherein the antigen-binding module comprisesa disulfide bond between a residue in the C_(H)1 domain and a residue inthe C_(L) domain.

Embodiment 3

The abTCR of embodiment 1 or 2, wherein the first polypeptide chainfurther comprises a first peptide linker between the firstantigen-binding domain and the first TCRD.

Embodiment 4

The abTCR of any one of embodiments 1-3, wherein the second polypeptidechain further comprises a second peptide linker between the secondantigen-binding domain and the second TCRD.

Embodiment 5

The abTCR of embodiment 3 or 4, wherein the first peptide linker and/orthe second peptide linker are, individually, from about 5 to about 50amino acids in length.

Embodiment 6

The abTCR of any one of embodiments 1-5, wherein the target antigen is acell surface antigen.

Embodiment 7

The abTCR of embodiment 6, wherein the cell surface antigen is selectedfrom the group consisting of protein, carbohydrate, and lipid.

Embodiment 8

The abTCR of embodiment 7, wherein the cell surface antigen is CD19,ROR1, ROR2, BCMA, GPRC5D, or FCRL5.

Embodiment 9

The abTCR of any one of embodiments 1-5, wherein the target antigen is acomplex comprising a peptide and a major histocompatibility complex(MHC) protein.

Embodiment 10

An abTCR that specifically binds to a target antigen, comprising:

a) a first polypeptide chain comprising a first antigen-binding domaincomprising a V_(H) antibody domain and a first TCRD comprising a firsttransmembrane domain of a first TCR subunit; and

b) a second polypeptide chain comprising a second antigen-binding domaincomprising a V_(L) antibody domains and a second TCRD comprising asecond transmembrane domain of a second TCR subunit,

wherein the V_(H) domain of the first antigen-binding domain and theV_(L) domain of the second antigen-binding domain form anantigen-binding module that specifically binds to the target antigen,

wherein the first TCRD and the second TCRD form a T cell receptor module(TCRM) that is capable of recruiting at least one TCR-associatedsignaling module, and

wherein the target antigen is a complex comprising a peptide and an MHCprotein.

Embodiment 11

The abTCR of embodiment 10, wherein the first polypeptide chain furthercomprises a first peptide linker between the first antigen-bindingdomain and the first TCRD and the second polypeptide chain furthercomprises a second peptide linker between the second antigen-bindingdomain and the second TCRD.

Embodiment 12

The abTCR of embodiment 11, wherein the first and/or second peptidelinkers comprise, individually, a constant domain or fragment thereoffrom an immunoglobulin or T cell receptor subunit.

Embodiment 13

The abTCR of embodiment 12, wherein the first and/or second peptidelinkers comprise, individually, a CH1, CH2, CH3, CH4 or CL antibodydomain, or a fragment thereof.

Embodiment 14

The abTCR of embodiment 12, wherein the first and/or second peptidelinkers comprise, individually, a Cα, Cβ, Cγ, or Cδ TCR domain, or afragment thereof.

Embodiment 15

The abTCR of any one of embodiments 1-14, wherein the first TCRD furthercomprises a first connecting peptide or fragment thereof of a TCRsubunit N-terminal to the first transmembrane domain.

Embodiment 16

The abTCR of any one of embodiments 1-15, wherein the second TCRDfurther comprises a second connecting peptide or fragment thereof of aTCR subunit N-terminal to the second transmembrane domain.

Embodiment 17

The abTCR of embodiment 15 or 16, wherein the TCRM comprises a disulfidebond between a residue in the first connecting peptide and a residue inthe second connecting peptide.

Embodiment 18

The abTCR of any one of embodiments 1-17, wherein the first TCRD furthercomprises a first TCR intracellular domain comprising a TCRintracellular sequence C-terminal to the first transmembrane domain.

Embodiment 19

The abTCR of any one of embodiments 1-18, wherein the second TCRDfurther comprises a second TCR intracellular domain comprising a TCRintracellular sequence C-terminal to the second transmembrane domain.

Embodiment 20

The abTCR of any one of embodiments 1-19, wherein the first polypeptidechain further comprises a first accessory intracellular domaincomprising a co-stimulatory intracellular signaling sequence C-terminalto the first transmembrane domain.

Embodiment 21

The abTCR of any one of embodiments 1-20, wherein the second polypeptidechain further comprises a second accessory intracellular domaincomprising a co-stimulatory intracellular signaling sequence C-terminalto the second transmembrane domain.

Embodiment 22

The abTCR of any one of embodiments 1-21, wherein the first polypeptidechain further comprises a first signaling peptide N-terminal to thefirst antigen-binding domain.

Embodiment 23

The abTCR of any one of embodiments 1-22, wherein the second polypeptidechain further comprises a second signaling peptide N-terminal to thesecond antigen-binding domain.

Embodiment 24

The abTCR of any one of embodiments 9-23, wherein the peptide in thetarget antigen complex is derived from a protein selected from the groupconsisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1,and PSA.

Embodiment 25

The abTCR of any one of embodiments 1-24, wherein the molecule binds tothe target antigen with an equilibrium dissociation constant (K_(d))from about 0.1 pM to about 500 nM.

Embodiment 26

The abTCR of any one of embodiments 1-25, wherein the TCR-associatedsignaling module is selected from the group consisting of CD3δε, CD3γε,and ζζ.

Embodiment 27

The abTCR of any one of embodiments 1-26, wherein the first TCR subunitis a TCR α chain, and the second TCR subunit is a TCR β chain.

Embodiment 28

The abTCR of any one of embodiments 1-26, wherein the first TCR subunitis a TCR β chain, and the second TCR subunit is a TCR α chain.

Embodiment 29

The abTCR of any one of embodiments 1-26, wherein the first TCR subunitis a TCR γ chain, and the second TCR subunit is a TCR δ chain.

Embodiment 30

The abTCR of any one of embodiments 1-26, wherein the first TCR subunitis a TCR δ chain, and the second TCR subunit is a TCR γ chain.

Embodiment 31

Nucleic acid(s) encoding the first and second polypeptide chains of theabTCR of any one of embodiments 1-30.

Embodiment 32

A complex comprising the abTCR of any one of embodiments 1-30 and atleast one TCR-associated signaling module selected from the groupconsisting of CD3δε, CD3γε, and ζζ.

Embodiment 33

The complex of embodiment 32, wherein the complex is an octamercomprising the abTCR and CD3δε, CD3γε, and ζζ.

Embodiment 34

An effector cell presenting on its surface the abTCR of any one ofembodiments 1-30 or the complex of embodiment 32 or 33.

Embodiment 35

An effector cell comprising the nucleic acid(s) of embodiment 31.

Embodiment 36

The effector cell of embodiment 34 or 35, wherein the effector cell doesnot express the first TCR subunit and/or the second TCR subunit.

Embodiment 37

The effector cell of embodiment 36, wherein

a) the first TCR subunit is TCRα and the second TCR subunit is TCRβ; or

b) the first TCR subunit is TCRβ and the second TCR subunit is TCRα; andwherein the effector cell is a γδ T cell.

Embodiment 38

The effector cell of embodiment 36, wherein

a) the first TCR subunit is TCRγ and the second TCR subunit is TCRδ; or

b) the first TCR subunit is TCRδ and the second TCR subunit is TCRγ; andwherein the effector cell is an αβ T cell.

Embodiment 39

The effector cell of any one of embodiments 34-36, wherein the effectorcell is modified to block or decrease the expression of a firstendogenous TCR subunit and/or a second endogenous TCR subunit.

Embodiment 40

The effector cell of embodiment 39, wherein

a) the first TCR subunit is TCRα and second TCR subunit is TCRβ; or

b) the first TCR subunit it TCRβ and the second TCR subunit is TCRα; and

wherein the effector cell is an αβ T cell modified to block or decreasethe expression of TCRα and/or TCRβ.

Embodiment 41

The effector cell of embodiment 39, wherein

a) the first TCR subunit is TCRγ and second TCR subunit is TCRδ; or

b) the first TCR subunit is TCRδ and the second TCR subunit is TCRγ; and

wherein the effector cell is a γδ T cell modified to block or decreasethe expression of TCRγ and/or TCRδ.

Embodiment 42

The effector cell of any one of embodiments 34-41, wherein the effectorcell is a CD3⁺ cell.

Embodiment 43

The effector cell of embodiment 42, wherein the CD3⁺ cell is selectedfrom the group consisting of a cytotoxic T cell, a helper T cell, anatural killer T cell, and a suppressor T cell.

Embodiment 44

The effector cell of any one of embodiments 34-43, comprising a) a firstvector comprising a first nucleic acid sequence encoding the firstpolypeptide chain of the abTCR under the control of a first promoter andb) a second vector comprising a second nucleic acid sequence encodingthe second polypeptide chain of the abTCR under the control of a secondpromoter.

Embodiment 45

The effector cell of any one of embodiments 34-43, comprising a vectorcomprising a) a first nucleic acid sequence encoding the firstpolypeptide chain of the abTCR under the control of a first promoter;and b) a second nucleic acid sequence encoding the second polypeptidechain of the abTCR under the control of a second promoter.

Embodiment 46

The effector cell of any one of embodiments 34-43, comprising a vectorcomprising a) a first nucleic acid sequence encoding the firstpolypeptide chain of the abTCR and a second nucleic acid sequenceencoding the second polypeptide chain of the abTCR, wherein the firstand second nucleic acid sequences are under the control of a singlepromoter.

Embodiment 47

The effector cell of any one of embodiments 34-45, wherein theexpression of the first polypeptide chain of the abTCR is more thantwo-fold different than the expression of the second polypeptide chainof the abTCR.

Embodiment 48

A method of killing a target cell presenting a target antigen,comprising contacting the target cell with the effector cell of any oneof embodiments 34-47, wherein the abTCR specifically binds to the targetantigen.

Embodiment 49

A method of killing a target cell presenting a target antigen,comprising contacting the target cell with an effector αβ T cellcomprising an abTCR that specifically binds to the target antigencomprising:

a) a first polypeptide chain comprising a first antigen-binding domaincomprising a V_(H) antibody domain and a first TCRD comprising a firsttransmembrane domain of a first TCR subunit; and

b) a second polypeptide chain comprising a second antigen-binding domaincomprising a V_(L) antibody domains and a second TCRD comprising asecond transmembrane domain of a second TCR subunit,

wherein the V_(H) domain of the first antigen-binding domain and theV_(L) domain of the second antigen-binding domain form anantigen-binding module that specifically binds to the target antigen,

wherein the first TCRD and the second TCRD form a T cell receptor module(TCRM) that is capable of recruiting at least one TCR-associatedsignaling module, and

wherein the first TCR subunit is TCRγ and the second TCR subunit isTCRδ, or the first TCR subunit is TCR and the second TCR subunit isTCRγ.

Embodiment 50

The method of embodiment 49, wherein the first polypeptide chain furthercomprises a first peptide linker between the first antigen-bindingdomain and the first TCRD and the second polypeptide chain furthercomprises a second peptide linker between the second antigen-bindingdomain and the second TCRD.

Embodiment 51

The method of embodiment 50, wherein the first and/or second peptidelinkers comprise, individually, a constant domain or fragment thereoffrom an immunoglobulin or T cell receptor subunit.

Embodiment 52

The method of embodiment 51, wherein the first and/or second peptidelinkers comprise, individually, a CH1, CH2, CH3, CH4 or CL antibodydomain, or a fragment thereof.

Embodiment 53

The method of embodiment 51, wherein the first and/or second peptidelinkers comprise, individually, a Cα, Cβ, Cγ, or Cδ TCR domain, or afragment thereof.

Embodiment 54

The method of any one of embodiments 48-53, wherein the contacting is invivo.

Embodiment 55

The method of any one of embodiments 48-53, wherein the contacting is invitro.

Embodiment 56

A pharmaceutical composition comprising the abTCR of any one ofembodiments 1-30, the nucleic acid(s) of embodiment 31, or the effectorcell of any one of embodiments 34-47, and a pharmaceutically acceptablecarrier.

Embodiment 57

A method of treating a target antigen-associated disease in anindividual in need thereof comprising administering to the individual aneffective amount of the pharmaceutical composition of embodiment 51.

Embodiment 58

A method of treating a target antigen-associated disease in anindividual in need thereof comprising administering to the individual aneffective amount of a composition comprising an effector αβ T cellcomprising an abTCR that specifically binds to the target antigencomprising:

a) a first polypeptide chain comprising a first antigen-binding domaincomprising a V_(H) antibody domain and a first TCRD comprising a firsttransmembrane domain of a first TCR subunit; and

b) a second polypeptide chain comprising a second antigen-binding domaincomprising a V_(L) antibody domains and a second TCRD comprising asecond transmembrane domain of a second TCR subunit,

wherein the V_(H) domain of the first antigen-binding domain and theV_(L) domain of the second antigen-binding domain form anantigen-binding module that specifically binds to the target antigen,

wherein the first TCRD and the second TCRD form a T cell receptor module(TCRM) that is capable of recruiting at least one TCR-associatedsignaling module, and

wherein the first TCR subunit is TCRγ and the second TCR subunit isTCRδ, or the first TCR subunit is TCRδ and the second TCR subunit isTCRγ.

Embodiment 59

The method of embodiment 58, wherein the wherein the first polypeptidechain further comprises a first peptide linker between the firstantigen-binding domain and the first TCRD and the second polypeptidechain further comprises a second peptide linker between the secondantigen-binding domain and the second TCRD.

Embodiment 60

The method of embodiment 59, wherein the first and/or second peptidelinkers comprise, individually, a constant domain or fragment thereoffrom an immunoglobulin or T cell receptor subunit.

Embodiment 61

The method of embodiment 60, wherein the first and/or second peptidelinkers comprise, individually, a CH1, CH2, CH3, CH4 or CL antibodydomain, or a fragment thereof.

Embodiment 62

The method of embodiment 60, wherein the first and/or second peptidelinkers comprise, individually, a Cα, Cβ, Cγ, or Cδ TCR domain, or afragment thereof.

Embodiment 63

The method of any one of embodiments 57-62, wherein the targetantigen-associated disease is cancer.

Embodiment 64

The method of embodiment 63, wherein the cancer is selected from thegroup consisting of adrenocortical carcinoma, bladder cancer, breastcancer, cervical cancer, cholangiocarcinoma, colorectal cancers,esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, headand neck cancer, kidney cancer, leukemia, lymphoma, lung cancer,melanoma, mesothelioma, multiple myeloma, pancreatic cancer,pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostatecancer, sarcoma, stomach cancer, uterine cancer and thyroid cancer.

Embodiment 65

The method of any one of embodiments 57-62, wherein the targetantigen-associated disease is viral infection.

Embodiment 66

The method of embodiment 65, wherein the viral infection is caused by avirus selected from the group consisting of Cytomegalovirus (CMV),Epstein-Barr Virus (EBV), Hepatitis B Virus (HBV), Kaposi's Sarcomaassociated herpesvirus (KSHV), Human papillomavirus (HPV), Molluscumcontagiosum virus (MCV), Human T cell leukemia virus 1 (HTLV-1), HIV(Human immunodeficiency virus), and Hepatitis C Virus (HCV).

Embodiment 67

A method of enriching a heterogeneous cell population for the effectorcell of any one of embodiments 34-47, comprising:

a) contacting the heterogeneous cell population with the target antigenimmobilized to a solid support to form complexes of the effector cellbound to the target antigen on the solid support; and

b) separating the complexes from the heterogeneous cell population,thereby generating a cell population enriched for the effector cell.

Embodiment 68

A nucleic acid library comprising sequences encoding a plurality ofabTCRs according to any one of embodiments 1-30.

Embodiment 69

A method of screening the nucleic acid library of embodiment 68 forsequences encoding abTCRs specific for a target antigen, comprising:

a) introducing the nucleic acid library into a plurality of CD3⁺ cells,such that the abTCRs are expressed on the surface of the plurality ofCD3⁺ cells;

b) incubating the plurality of CD3⁺ cells with labelled target antigen;

c) collecting CD3⁺ cells bound with the labelled target antigen; and

d) isolating sequences encoding the abTCRs from cells collected in stepc), thereby identifying abTCRs specific for the target antigen.

EXAMPLES

Materials and Methods

Cell Samples, Cell Lines, and Antibodies

The cell lines HepG2 (ATCC HB-8065; HLA-A2+, AFP+), SK-HEP-1 (ATCCHTB-52; HLA-A2+, AFP⁻), Raji (ATCC CCL-86; CD19⁺), CA46 (ATCC CRL-1648;CD19⁺), Jurkat (ATCC CRL-2899, CD19⁻), J.RT3-T3.5 (ATCC TIB-153), Jeko-1(ATCC CRL-3006; CD19⁺), THP-1 (ATCC TIB-202, CD19⁻), Daudi (ATCCCCL-213; CD19⁺), HeLa (ATCC CCL-2), MDA-MB-231 (ATCC HTB-26) and MCF-7(ATCC HTB-22) were obtained from the American Type Culture Collection.Jurkat is a human T lymphocyte cell line derived from T cell leukemia.J.RT3-T3.5 is a mutant line derived from Jurkat cells that lacks the Tcell receptor β chain. Raji is a Burkitt lymphoma cell line thatexpresses CD19. Raji-CD19 knockout (Raji-CD19KO) line was generated byCRISPR technology. Three different guide sequences were designed totarget CD19 in Raji cells. CRISPR-Cas9 vector was purchased from Origeneand each guide was cloned separately into the pCas-Guide vector. Threedays after electroporation, efficiency of knock-out by each guide wasevaluated by flow cytometry and the best CD19-knock-out pool was chosenfor clonal selection by limiting dilution. The selected clone wasconfirmed as a complete CD19 knock-out by sequencing. Another controlcell line, SK-HEP-1-AFP-MG was generated by transducing SK-HEP-1 cellline with a minigene cassette expressing an AFP peptide AFP158 (SEQ IDNO: 53), which results in a high level of cell surface expression ofAFP158/HLA-A*02:01 complex. All cell lines were cultured in RPMI 1640 orDMEM supplemented with 10% FBS and 2 mM glutamine at 37° C./5% CO₂.

Monoclonal Ab against human HLA-A02 (clone BB7.2) conjugated to FITC orAPC, and its isotype control mouse IgG 2b conjugated to FITC or APC,antibodies against human or mouse CD3, human T cell receptor varioussubunit, 3×Flag tag, HA tag, goat F(ab)2 anti-human IgG conjugated withPE or FITC, and fluorescence-conjugated goat F(ab′)2 anti-mouse Ig's(Invitrogen) were purchased. The anti-idiotypic antibody against anAFP158/HLA-A*02:01-specific antibody was developed and produced in houseat Eureka Therapeutics. Flow cytometry data were collected using BDFACSCanto II and analyzed using FlowJo software package.

All peptides were purchased and synthesized by Elim Biopharma. Peptideswere >90% pure. The peptides were dissolved in DMSO or diluted in salineat 10 mg/mL and frozen at −80° C. Biotinylated single chainAFP158/HLA-A*02:01 and control peptides/HLA-A*02:01 complex monomerswere generated by refolding the peptides with recombinant HLA-A*02:01and beta-2 microglobulin (β2M). The monomers were biotinylated via theBSP peptide linked to the C-terminal end of HLA-A*02:01 extracellulardomain (ECD) by the BirA enzyme. Fluorescence-labelled streptavidin wasmixed with biotinylated peptide/HLA-A*02:01 complex monomer to formfluorescence-labelled peptide/HLA-A*02:01 tetramer.

Lentiviruses containing human CD19-specific orAFP158/HLA-A*02:01-specific CAR or abTCRs were produced, for example, bytransfection of 293T cells with vectors encoding the chimericconstructs. Primary human T-cells were used for transduction afterone-day stimulation with CD3/CD28 beads (Dynabeads®, Invitrogen) in thepresence of interleukin-2 (IL-2) at 100 U/ml. Concentrated lentiviruseswere applied to T-cells in Retronectin- (Takara) coated 6-well platesfor 96 hours. Transduction efficiencies of the anti-AFP and anti-CD19chimeric constructs were assessed by flow cytometry, using biotinylatedAFP158/HLA-A*02:01 tetramer (“AFP158 tetramer”) with PE-conjugatedstreptavidin or anti-myc antibody respectively. Repeat flow cytometryanalyses were done on day 5 and every 3-4 days thereafter.

Cell lines were transduced with either one or two vectors that encodethe two subunits of abTCR construct. Five days post-transduction, celllysates were generated for western blot using anti-HA (Anti-HA tagantibody—ChIP Grade, Abcam) or anti-Flag antibody (Anti-Flag AntibodyProduced in Rabbit, Sigma).

Tumor cytotoxicities were assayed by Cytox 96 Non-radioactive LDHCytotoxicity Assay (Promega). CD3⁺ T cells were prepared fromPBMC-enriched whole blood using EasySep Human T Cell Isolation Kit(StemCell Technologies) which negatively depletes CD14, CD16, CD19,CD20, CD36, CD56, CD66b, CD123, glycophorin A expressing cells. Human Tcells were activated and expanded with, for example, CD3/CD28 Dynabeads(Invitrogen) according to manufacturer's protocol. Activated T cells(ATC) were cultured and maintained in RPMI1640 medium with 10% FBS plus100 U/ml IL-2, and used at day 7-14. Activated T cells (effector cells)and target cells were co-cultured at various effector-to-target ratios(e.g., 2.5:1 or 5:1) for 16 hours and assayed for cytotoxicities.

Example 1. Antibody-T Cell Receptor (abTCR) Chimera Designs

Four different antibody-T cell receptor chimeric construct (abTCRs)designs (abTCR-3, abTCR-4, abTCR-5, and abTCR-6), including contemplatedvariations, are shown in FIGS. 1A and 1B. In these designs, the heavy(IgV_(H)-IgC_(H)1) and light (IgV_(L)-IgC_(L)) chain domains of anantibody Fab fragment are fused to the amino terminus of T cell receptorα/β chain or γ/δ chain fragments lacking variable and constant domainsand including all or part of their connecting peptide (region after theconstant domain) to form chimeric antibody-TCR heterodimers which can beexpressed on the surface of T cells. The IgV_(H) and IgV_(L) domains ineach of the abTCR designs determine the antigen-binding specificity, andtogether with the IgC_(H)1 and IgC_(L), form a structure that resemblesa Fab fragment. In a native TCR, the Vα/Vβ or Vδ/Vγ domains form theantigen-binding domain of the TCR. These designs replace the Vα-Cα/Vβ-Cβor Vδ-Cδ/Vγ-Cγ regions with IgV_(H)-IgC_(H)1 or IgV_(L)-IgC_(L), thusconferring an antibody's binding specificity to the construct, whilemaintaining the ability of the construct to be associated with theaccessory molecules in a native TCR complex, such as CD3δε, CD3γε andCD3ζζ. These designs are distinct from the cTCR designs described byGross and Eshhar (Endowing T cells with antibody specificity usingchimeric T cell receptors, FASEB J. 1992 (15):3370), where the variabledomains of antibodies are linked to TCR constant regions, replacing onlythe Vα/Vβ regions with IgV_(H)/IgV_(L).

In other abTCR designs, the heavy (IgV_(H)) and light (IgV_(L)) chaindomains of an antibody Fv fragment that is specific for a complexcomprising a peptide and an MHC protein (an MHC-restricted antibodymoiety) are fused to the amino terminus of T cell receptor α/β chain orγ/δ chain fragments lacking variable domains and including all or partof their connecting peptides (region after the constant domain). In someof these abTCR designs, the T cell receptor α/β chain or γ/δ chainfragments include all or part of the TCR constant domains. In one suchdesign, abTCR-7, the IgV_(H) is fused to a TCRδ fragment including theconstant domain and the IgV_(L) is fused to a TCRγ fragment includingthe constant domain. These designs are distinct from the cTCR designsdescribed by Gross and Eshhar (supra), where the antibody variabledomains are for non-MHC-restricted binding.

In the abTCR-3 (IgV_(H)-IgC_(H)1-TCRα/IgV_(L)-IgC_(L)-TCRβ) design, thevariable domain and the first constant domain (IgV_(H)-IgC_(H)1) of anantibody heavy chain replaces the amino terminal portion of the TCRαchain up to a position bordering or within the connecting peptide in theextracellular domain after the Vα-Cα region. The variable domain and theconstant domain (IgV_(L)-IgC_(L)) of the corresponding antibody lightchain replaces the amino terminal portion of the TCRβ chain up to aposition bordering or within the connecting peptide in the extracellulardomain after the Vβ-Cβ region. In the abTCR-4(IgV_(H)-IgC_(H)1-TCRβ/IgV_(L)-IgC_(L)-TCRα) design, the variable domainand the first constant domain (IgV_(H)-IgC_(H)1) of an antibody heavychain replaces the amino terminal portion of the TCRβ chain up to aposition bordering or within the connecting peptide in the extracellulardomain after the Vβ-Cβ region. The variable domain and the constantdomain (IgV_(L)-IgC_(L)) of the corresponding antibody light chainreplaces the amino terminal portion of the TCRα chain up to a positionbordering or within the connecting peptide in the extracellular domainafter the Vα-Cα region. The chimeric α and β chains are dimerizedthrough two disulfide bonds, one between the IgC_(L) and the IgC_(H)1domains, and one between the connecting peptides in the TCRα and βchains. A 3×-Flag tag is optionally fused to the C-terminus of the TCRαchain cytoplasmic region, and an HA tag is optionally fused to theC-terminus of the TCRβ chain cytoplasmic region.

In one abTCR-3 embodiment, one chain includes the sequence of SEQ ID NO:23 (anti-AFP158/HLA-A*02:01-abTCR-3), where the IgV_(H) domain of ananti-AFP158/HLA-A*02:01 antibody (SEQ ID NO: 38) is fused to an IgC1ldomain (SEQ ID NO: 39) fused to SEQ ID NO: 15, a portion of the TCRαchain including part of the connecting peptide in the extracellulardomain of the TCRα chain after the Vα-Cα region, and the other chainincludes the sequence of SEQ ID NO: 24, where the IgV_(L) domain of theanti-AFP158/HLA-A*02:01 antibody (SEQ ID NO: 40) is fused to an IgC_(L)domain (SEQ ID NO: 41) fused to SEQ ID NO: 16, a carboxy portion of theTCRβ chain including part of the connecting peptide in the extracellulardomain of the TCRβ chain after the Vβ-Cβ region. In one abTCR-4embodiment, one chain includes the sequence of SEQ ID NO: 25(anti-AFP158/HLA-A*02:01-abTCR-4), where the IgV_(L) domain of ananti-AFP158/HLA-A*02:01 antibody (SEQ ID NO: 40) is fused to an IgC_(L)domain (SEQ ID NO: 41) fused to SEQ ID NO: 15, a portion of the TCRαchain including part of the connecting peptide in the extracellulardomain of the TCRα chain after the Vα-Cα region, and the other chainincludes the sequence of SEQ ID NO: 26, where the IgV_(H) domain of theanti-AFP158/HLA-A*02:01 antibody (SEQ ID NO: 38) is fused to an IgC_(H)1domain (SEQ ID NO: 39) fused to SEQ ID NO: 16, a carboxy portion of theTCRβ chain including part of the connecting peptide in the extracellulardomain of the TCRβ chain after the Vβ-Cβ region.

In the abTCR-5 (IgV_(H)-IgC_(H)1-TCRγ/IgV_(L)-IgC_(L)-TCRδ) design, thevariable domain and the first constant domain (IgV_(H)-IgC_(H)1) of anantibody heavy chain replaces the amino terminal portion of the TCRγchain up to a position bordering or within the connecting peptide in theextracellular domain of the TCRγ chain after the Vγ-Cγ region. Thevariable domain and the constant domain (IgV_(L)-IgC_(L)) of thecorresponding antibody light chain replaces the amino terminal portionof the TCRδ chain up to a position bordering or within the connectingpeptide in the extracellular domain of the TCRδ chain after the Vδ-Cδregion. In the abTCR-6 (IgV_(H)-IgC_(H)1-TCRδ/IgV_(L)-IgC_(L)-TCRγ)design, the variable domain and the first constant domain(IgV_(H)-IgC_(H)1) of an antibody heavy chain replaces the aminoterminal portion of the TCRδ chain up to a position bordering or withinthe connecting peptide in the extracellular domain of the TCRδ chainafter the Vδ-Cδ region. The variable domain and the constant domain(IgV_(L)-IgC_(L)) of the corresponding antibody light chain replaces theamino terminal portion of the TCRγ chain up to a position bordering orwithin the connecting peptide in the extracellular domain of the TCRγchain after the Vγ-Cγ region. The chimeric γ and δ chains are dimerizedthrough two disulfide bonds, one between the IgC_(L) and the IgC_(H)1domains, and one between the connecting peptides in the TCRγ and δchains. A 3×flag tag is optionally fused to the C-terminus of the TCRγchain cytoplasmic region, and an HA tag is optionally fused to theC-terminus of the TCRδ chain cytoplasmic region.

In one abTCR-5 embodiment, one chain includes the sequence of SEQ ID NO:30 (anti-AFP158/HLA-A*02:01-abTCR-5), where the IgV_(H) domain of ananti-AFP158/HLA-A*02:01 antibody (SEQ ID NO: 38) is fused to an IgC_(H)1domain (SEQ ID NO: 39) fused to SEQ ID NO: 20, a portion of the TCRγchain including part of the connecting peptide in the extracellulardomain of the TCRγ chain after the Vγ-Cγ region, and the other chainincludes the sequence of SEQ ID NO: 29, where the IgV_(L) domain of theanti-AFP158/HLA-A*02:01 antibody (SEQ ID NO: 40) is fused to an IgC_(L)domain (SEQ ID NO: 41) and then to SEQ ID NO: 19, a carboxy portion ofthe TCRδ chain including part of the connecting peptide in theextracellular domain of the TCRδ chain after the Vδ-Cδ region. In oneabTCR-6 embodiment, one chain includes the sequence of SEQ ID NO: 34(anti-AFP158/HLA-A*02:01-abTCR-6) where the IgV_(L) domain of ananti-AFP158/HLA-A*02:01 antibody (SEQ ID NO: 40) is fused to an IgC_(L)domain (SEQ ID NO: 41) fused to SEQ ID NO: 20, a portion of the TCRγchain including part of the connecting peptide in the extracellulardomain of the TCRγ chain after the Vγ-Cγ region, and the other chainincludes the sequence of SEQ ID NO: 33, where the IgV_(H) domain of theanti-AFP158/HLA-A*02:01 antibody (SEQ ID NO: 38) is fused to an IgC_(H)1domain (SEQ ID NO: 39) fused to SEQ ID NO: 19, a carboxy terminalportion of the TCRδ chain including part of the connecting peptide inthe extracellular domain of the TCRδ chain after the Vδ-Cδ region.

As illustrated in FIG. 1B, variations of each of the four abTCR designsare also contemplated. Such variations may include varying the length ofthe extracellular domain, such as (i) lengthening by adding residues atthe junction formed by the IgC and TCR fusion or (ii) shortening bydeleting residues at the N-terminal of the TCR connecting peptides. Anembodiment of such a variation of abTCR-6 is abTCR-6MD, where one chainincludes the sequence of SEQ ID NO: 36(anti-AFP158/HLA-A*02:01-abTCR-6MD), where the IgV_(L) domain of ananti-AFP158/HLA-A*02:01 antibody (SEQ ID NO: 40) is fused to an IgC_(L)domain (SEQ ID NO: 41) fused to SEQ ID NO: 22, a carboxy terminalportion of the TCRγ chain including a longer (compared to abTCR-6)portion of the connecting peptide after the Vγ-Cγ region in the TCRγchain, and the other chain includes the sequence of SEQ ID NO: 35, wherethe IgV_(H) domain of the anti-AFP158/HLA-A*02:01 antibody (SEQ ID NO:38) is fused to an IgC_(H)1 domain (SEQ ID NO: 39) fused to SEQ ID NO:21, a carboxy terminal portion of the TCRδ chain including a longer(compared to abTCR-6) portion of the connecting peptide after the Vδ-Cδregion of the TCRδ chain. An embodiment of such a variation of abTCR-5is abTCR-5MD, where one chain includes the sequence of SEQ ID NO: 31(anti-AFP158/HLA-A*02:01-abTCR-5MD) where the IgV_(L) domain of theanti-AFP158/HLA-A*02:01 antibody (SEQ ID NO: 40) is fused to an IgC_(L)domain (SEQ ID NO: 41) fused to SEQ ID NO: 21, a carboxy terminalportion of the TCRδ chain including a longer (compared to abTCR-5)portion of the connecting peptide after the Vδ-Cδ region of the TCRδchain, and the other chain includes the sequence of SEQ ID NO: 32, wherethe IgV_(H) domain of an anti-AFP158/HLA-A*02:01 antibody (SEQ ID NO:38) is fused to an IgC_(H)1 domain (SEQ ID NO: 39) fused to SEQ ID NO:22, a carboxy terminal portion of the TCRγ chain including a longer(compared to abTCR-5) portion of the connecting peptide after the Vγ-Cγregion in the TCRγ chain. An embodiment of such a variation of abTCR-4is abTCR-4MD, where one chain includes the sequence of SEQ ID NO: 27(anti-AFP158/HLA-A*02:01-abTCR-4MD) where the IgV_(L) domain of ananti-AFP158/HLA-A*02:01 antibody (SEQ ID NO: 40) is fused to an IgC_(L)domain (SEQ ID NO: 41) fused to SEQ ID NO: 17, a carboxy terminalportion of the TCRα chain including a longer (compared to abTCR-4)portion of the connecting peptide after the Vα-Cα region, and the otherchain includes the sequence of SEQ ID NO: 28, where the IgV_(H) domainof the anti-AFP158/HLA-A*02:01 antibody (SEQ ID NO: 38) is fused to anIgC_(H)1 domain (SEQ ID NO: 39) fused to SEQ ID NO: 18, a carboxyterminal portion of the TCRβ chain including a longer (compared toabTCR-4) portion of the connecting peptide after the Vβ-Cβ region.

Additional variations may include fusing additional effector domains(e.g., intracellular domain of CD28) to the C-terminal end of any of theTCRα/β/δ/γ chains. Another variation may include varying the linkerregion between the IgV and IgC domains.

Example 2: Expression of abTCRs in T Cell Lines

In mature T cells, the TCR-CD3 complex is composed of four dimericmodules: TCRαβ (or TCRγ6), CD3δε, CD3γε and CD3ζζ, which is thought toassociate through intramembrane and extramembrane contacts to form theintact complex, as shown in FIG. 2 (from Wucherpfennig K W, et al.,Structural biology of the T-cell receptor: insights into receptorassembly, ligand recognition, and initiation of signaling. Cold SpringHarb Perspect Biol. 2010 April; 2(4):a005140). Complex assembly occursin the endoplasmic reticulum (ER). Only complete TCR-CD3 complexes aretransferred into the Golgi apparatus where they go through theglycosylation process and get transported to the plasma membrane of Tcells. Incomplete TCRs are directed from the Golgi to the lysosomes,where they are degraded.

To test abTCR expression in T cells and to examine whether abTCRs canfunction like endogenous TCRs in recruiting CD3 molecules and enablingthe expression of the abTCR-CD3 complex on T cell surface, abTCRconstructs were introduced into a mutant Jurkat T cell line, J.RT3-T3.5.Unlike Jurkat, an αβ TCR-positive leukemia T cell line, J.RT3-T3.5 is aJurkat mutant line which lacks TCR β subunit expression. Since theassembly of TCR-CD3 complex is impaired without the TCR β subunit,neither TCR nor CD3 can be transported to the plasma membrane inJ.RT3-T3.5 cells.

Detection of abTCR Expression by Western Blot

Five sets of abTCR constructs (abTCR-3, -4, -5, -6, -6MD) were generatedwith the IgV_(H) and IgV_(L) regions of an anti-AFP158/HLA-A*02:01antibody. J.RT3-T3.5 and Jurkat cells were transduced with abTCR-3 (SEQID NOs: 23 and 24), abTCR-4 (SEQ ID NOs: 25 and 26), abTCR-5 (SEQ IDNOs: 29 and 30), abTCR-6 (SEQ ID NOs: 33 and 34), or abTCR-6MD (SEQ IDNOs: 35 and 36) constructs and expression of individual subunits ofabTCRs was detected in western blots using anti-Flag or anti-HAantibodies (FIG. 3). For each construct, the two subunits were subclonedinto two separate lentiviral vectors. To express the complete abTCRheterodimer, T cells were transduced with both vectors. TCRβ and TCRδchimeras were tagged with HA while TCRα and TCRγ chimeras were taggedwith 3×Flag attached at the C-termini of the abTCR subunits. The TCRchains bearing HA- or 3×Flag-tags are indicated in parenthesis in FIG.3, under the label for each abTCR design.

Among the HA-tagged chimeras in both J.RT3-T3.5 and Jurkat (FIG. 3,anti-HA panels), the IgV_(L)-IgC_(L)-TCRβ subunit in abTCR-3 exhibitedthe highest expression, followed by the IgV_(H)-IgC_(L)-TCRδ subunit inabTCR-6 and abTCR-6MD and theIgV_(H)-IgC_(H)1-TCRβ subunit in abTCR-4.Among the 3×Flag-tagged chimeras (FIG. 3, anti-flag panels), the highestexpression was observed for IgV_(L)-IgC_(L)-TCRγ in abTCR-6 andabTCR-6MD, followed by IgV_(L)-IgC_(L)-TCRα in ab-TCR-4. Both chains forabTCR-5 TCRγ/IgV_(L)-IgC_(L)-TCRδ) exhibited the lowest expression amongthe 5 sets of constructs tested. The TCRδ chain for abTCR-6MD wasexpressed at a similar level as abTCR6, while the TCRγ chain forabTCR-6MD was expressed at a lower level than observed for abTCR6. Boththe percentage of cells transduced and the level of expression withinthe transduced cells contribute to the signals detected in westernblots. Therefore, flow cytometry was next performed to determine thelevel of abTCR expression on the cell surface.

Detection of abTCR Cell-Surface Expression and TCR-CD3 Complex Formationby Flow Cytometry

The 5 pairs of chimeric abTCR chains described above (abTCR-3, -4, -5,-6, -6MD) were individually transduced into J.RT3-T3.5 (FIGS. 4A-4C) andJurkat (FIGS. 5A-5C) cells. The cells transduced with abTCR constructswere assessed by the following: (i) anti-CD3ε antibody to assess therescue of CD3ε expression on J.RT3-T3.5 cells (FIG. 4A), (ii) anti-TCRαβantibody to assess the impact of abTCR constructs on endogenousexpression of TCRαβ in Jurkat cells (FIG. 5A), (iii) PE-labelledAFP158/HLA-A*02:01 tetramer to assess antigen binding by the transducedabTCR constructs (FIGS. 4B and 5B) and (iv) anti-idiotype antibodyagainst the anti-AFP158/HLA-A*02:01 antibody used in the abTCR chimeras(FIGS. 4C and 5C) to assess the surface expression of the chimericconstructs.

For J.RT3-T3.5 cells, mock transduction did not confer binding toAFP158/HLA-A*02:01 tetramers and anti-idiotype antibody and did notresult in CD3ε expression on the cell surface (FIGS. 4A-4C).Anti-idiotype antibody detected a shoulder extending from theabTCR-negative peak in J.RT3-T3.5 cells transduced with abTCR-3 andabTCR-4. In contrast, cells transduced with abTCR-5, -6 and -6MDdisplayed distinct peaks at high fluorescence intensities when stainedwith the anti-idiotype antibody (FIG. 4C). The abTCR constructs arefunctional in being able to bind target antigen AFP158 tetramer (FIG.4B). A larger population of cells expressed abTCR-6MD constructscompared to abTCR-6, as evident by a higher AFP158 tetramer-positivepeak in abTCR-6MD. However, the abTCR-6MD transduced cells appear toexpress a similar copy number per cell as abTCR-6 transduced cells,since the AFP158 tetramer-positive peaks have similar mean fluorescenceintensity (MFI). Additionally, expression of abTCR constructs rescuedcell surface expression of CD3ε in J.RT3-T3.5 cells (FIG. 4A). This isunexpected since the constant domains of the TCR have been attributed tothe interaction with the CD3 chains (reviewed by Kuhns and Davis, TCRSignaling Emerges from the Sum of Many Parts, Front Immunol. 2012; 3:159, Wang and Reinherz, The structural basis of αβ T-lineage immunerecognition: TCR docking topologies, mechanotransduction, andco-receptor function, Immunol Rev. 2012, 250:102). Since the chimerasreplaced the TCR constant domains with IgC, we demonstrated that the TCRconstant domain is not necessary for CD3 assembly with the TCR complex.When the abTCR-6 and abTCR-6MD transduced cells were co-stained withanti-CD3ε and AFP158/HLA-A*02:01 tetramers and analyzed by flowcytometry, we confirmed that the CD₃ε⁺ J.RT3-T3.5 cells are also AFP158tetramer-positive (FIG. 6). This indicates that the exogenous abTCRchimeras form functional receptors that can bind their cognate antigens,and rescue the cell surface expression of CD3 complex on J.RT3-T3.5cells.

The same set of experiments were also conducted in Jurkat cells (an αβTCR positive T cell line), using the abTCR-3, -4, -5, -6 and -6MDconstructs with the anti-AFP158/HLA-A*02:01 antibody (FIG. 5A-5C). Theresults are consistent with that observed in J.RT3-T3.5 cells in termsof AFP158 tetramer staining (FIG. 5B) and anti-idiotypic antibodybinding (FIG. 5C). The transduced cells were also stained with ananti-TCRα/β antibody to determine the impact of the abTCR constructs onthe expression of endogenous TCRα/β chains. While mock-transduced Jurkatcells expressed a high level of TCRα/β, a TCRα/β negative population wasdetected in each of abTCR-transduced cells as a shoulder on the left ofthe TCRα/β peak (FIG. 5A). These data suggest that expression of theabTCR chimeras competes for the CD3 chains, resulting in a reduction inthe surface expression of endogenous TCRα/β.

Combining the observations from the western blots and flow cytometryexperiments, we postulate that in abTCR-3 and -4 transduced cells, someof the endogenous TCRα subunit may pair with the exogenous β chains ofthe abTCR chimera, to form TCR-CD3 complexes that can be transported tothe cell surface. Alternatively, abTCR-3 and -4 may have differentconformation which limited the exposure of the epitope for theanti-idiotype antibody. In abTCR-3-transduced J.RT3-T3.5 and Jurkatcells, the high level expression of the IgV_(L)-IgC_(L)-TCRβ chain (perwestern blot, FIG. 3) resulted in a large percentage of J.RT3-T3.5 cellsthat express CD3ε on the cell surface (FIG. 4A) and a reduction inendogenous TCRα/β expression in a subset of the Jurkat cells transducedwith abTCR-3. In abTCR-4 transduced cells, the IgV_(H)-IgC_(H)1-TCRβchain also resulted in CD3ε expression in J.RT3-T3.5 cells and reductionin endogenous TCRα/β expression in Jurkat cells, but to a much lesserextent since the chimeric abTCR β chain expression is much lower inabTCR-4 compared to abTCR-3 (per western blot, FIG. 3).

For abTCR-3 and -4 transduced cells, the pairing of exogenous TCRβchimera with the endogenous TCRα chains in TCRαβ⁺ T cells is expected toreduce the pool of TCRβ chimera chains available for correct pairingwith exogenous TCRα chimera chains. This will not be an issue in TCRαβ⁺T cells when expressing abTCR-5, -6 or -6MD constructs, where thechimeras are generated with the TCRδ and TCRγ chains. The high MFI inthe abTCR positive peaks are consistent with a high number ofcorrectly-paired chimeras in both J.RT3-T3.5 and Jurkat cells thatexpress the abTCR-5, -6 or -6MD constructs. Conversely, usage of abTCR-3and -4 constructs, where the chimeras are generated with the TCRα andTCRβ chains, for expression in TCRδγ⁺ T cells would be preferred toavoid the pairing of the exogenous chimeric chains with the endogenousTCRδ and TCRγ chains.

Example 3. Expression of abTCR in Primary T Cells

Having demonstrated that the abTCR constructs can be successfullytransduced into T cell lines and expressed on the cell surface alongwith CD3 complex as functional antigen-binding receptors, we next testedthe expression of abTCR in primary T cells.

abTCR Expressed in CD4+ and CD8+ Primary T Cells

Peripheral blood lymphocytes were isolated from healthy donors andtransduced with an abTCR-6MD construct encoding ananti-AFP158/HLA-A*02:01 binding moiety (SEQ ID NOs: 35 and 36). TheabTCRγ and δ subunits were subcloned into the same lentiviral vector totransduce primary human T cells. After 5 days of transduction, abTCR-Tcells and mock-transduced cells were co-stained with AFP158 tetramer,and CD4 and CD8 antibodies and analyzed by flow cytometry. FIG. 7A showsa scatter plot of CD8 vs antigen (AFP158 tetramer) binding, while FIG.7B shows scatter plots of CD8 vs CD4. In the mock-transduced T cells,the CD4:CD8 ratio is about 2:1 (FIG. 7B top panel). The same CD4:CD8ratio was observed in cells transduced with the abTCR-6MD construct(FIG. 7B middle panel). We found that CD4:CD8 ratio is also about 2:1among the AFP158 tetramer⁺ population in the abTCR-6MD transduced cells(see FIG. 7B bottom panel and gating in FIG. 7A). This indicates thatthe abTCR chimera can be expressed in both CD4⁺ and CD8⁺ primary Tcells.

Exogenous abTCR Chains are Physically Associated with the CD3 Complex

Given that abTCR expression in T cell lines was able to rescue surfaceexpression of CD3ε in J.RT3-T3.5 cells, we tested if abTCR constructsexpressed in primary T cell are physically associated with individualchains in the CD3 complex by co-immunoprecipitation (co-ip). Primary Tcells were stimulated using anti-CD3 and anti-CD28 and thenmock-transduced or transduced with abTCR-6MD constructs encoding ananti-AFP158/HLA-A*02:01 binding moiety (SEQ ID NOs: 35 and 36). Twelvedays after transduction, abTCR-T cells were co-cultured withSK-HEP-1-AFP-MG for another 12 days to enrich for AFP158 tetramer+cells. The cells were then lysed with digitonin (0.1%) lysis buffer andan anti-Flag antibody was used to i.p. the TCRγ chain via the 3×Flagtag. As shown in FIG. 8, the CD3δ, CD3ε, CD3γ and CD3ζ chains wereco-immunoprecipitated with abTCRγ chimera, demonstrating that thetransduced abTCR chimeras were physically associated with the endogenousCD3 complex. The band with a higher MW than CD3ε, observed in themock-transduced sample with anti-Flag immunoprecipitation, is anon-specific band.

Similar co-immunoprecipitation experiments are done in JRT3-T3.5 andJurkat cell lines. JRT3-T3.5 and Jurkat cell lines are transduced withabTCR-6MD constructs encoding an anti-AFP158/HLA-A*02:01 binding moiety(SEQ ID NOs: 35 and 36). 5 days post-transduction, CELLection biotinbinder kit is used to purify AFP158 tetramer+ populations from JRT3-T3.5and Jurkat cell lines. Jurkat and JRT3-T3.5 cells transduced withabTCR-6MD and purified with AFP158 tetramer are named asJurkat-abTCR-pure and JRT3-T3.5-abTCR-pure, respectively. JRT3-T3.5,JRT3-T3.5-abTCR-pure, Jurkat and Jurkat-abTCR-pure cells are expandedand lysed in 0.1% digitonin lysis buffer. Co-immunoprecipitation isperformed using anti-Flag antibody (Sigma) and Dynabeads Protein G forimmunoprecipitation (Life Technologies) following standard protocol.

Example 4. Characterizing Biological Activities of T Cells Transducedwith abTCR-6MD and CAR Constructs Containing the SameAnti-AFP158/HLA-A*02:01 Variable Domains

abTCR-Transduced T Cells can Specifically Kill Antigen Positive CancerCells

Primary T cells were mock-transduced or transduced with lentiviralvectors encoding a CAR containing an anti-AFP158/HLA-A*02:01 scFv (SEQID NO: 37) or an abTCR-6MD containing the same anti-AFP158/HLA-A*02:01variable domains (SEQ ID NOs: 35 and 36). The transduction efficiencywas determined by staining with PE-labeled AFP158/HLA-A*02:01 tetramers(FIG. 9A). T cell populations with similar transduction rates (32% forCART and 34% for abTCR) were used to test their abilities to kill cancercell lines. Three cell lines were used: HepG2 (AFP+/HLA-A2+), SK-HEP-1(AFP−/HLA-A2+) and SK-HEP-1-AFP-MG (SK-HEP-1 transduced with an AFPminigene) at an effector-to-target ratio of 2.5:1. Specific lysis wasmeasured after 16 hr incubation using the Cytox 96 Non-radioactiveCytotoxicity Assay (Promega). As shown in FIG. 9B, T cells transducedwith both CAR and abTCR-6MD bearing an anti-AFP158/HLA-A*02:01 bindingmoiety directed killing of antigen-positive cell lines HepG2 andSK-HEP-1-AFP-MG, but did not lead to killing of antigen-negative cellline SK-HEP-1. The level of specific lysis observed in abTCR-transducedcells is equivalent to that for CAR-T cells.

abTCR-Transduced T Cells Degranulate Upon Antigen Stimulation

To further characterize the biological activities in abTCR- vsCAR-transduced T cells, we used a flow cytometry assay to detect CD107asurface expression as a measurement of degranulation activity. abTCR-and CAR-transduced T cells with an anti-AFP158/HLA*02:01 binding moietywere generated as above and were co-incubated with HepG2, SK-HEP-1 andSK-HEP-1-AFP-MG cells for 4 hours in the presence of a 1:200 dilution ofanti-CD107a antibody and protein transport inhibitor cocktail(eBioscience). After co-incubation with target cells, transduced T cellswere stained with AFP158/HLA tetramers and anti-CD8. Degranulation intetramer-positive, CD8-positive T cells is shown in FIG. 10, rightpanel. The highest level of degranulation, as measured by CD107aexpression, was observed upon co-incubation with SK-HEP-1-AFP-MG (solidline, gray-filled), followed by HepG2 (dotted line, gray-filled), whileno degranulation was observed with the parental antigen-negativeSK-HEP-1 (solid line, white-filled) with both abTCR and CAR-transduced Tcells. The level of degranulation was similar between abTCR- andCAR-transduced cells when the same target cells were used. This isconsistent with the T-cell mediated cell lysis data above. Takentogether, we demonstrated that abTCR-transduced T cells are equally asresponsive as CAR-transduced cells to antigen-positive cancer cells indegranulation (FIG. 10) and mediating cell killing (FIG. 9).

Cytokine Production and Secretion by abTCR and CAR T Cells in Tumor CellKilling

T cells transduced with either abTCR or CAR and having similartransduction rates were generated and co-incubated with target cells asabove. Release of IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, IFN-γ and TNF-αinto the media after the in vitro killing assay shown in FIG. 9B wasmeasured using the Magpix multiplex system (Luminex) with the Bio-plexPro Human Cytokine 8-plex Assay (BioRad). To reach the detection limitsof the assay supernatants from SK-HEP-1-AFP-MG target reactions werediluted 25-fold, while all other samples were undiluted. Cytokineconcentrations were determined with a known standard curve, aftersubtracting cytokine release from media, target alone and effecteralone.

We estimated the number of AFP158/HLA-A*02:01 complexes on the surfaceof HepG2 to be ˜100 per cell, using high resolution microscopy (data notshown). At such a low copy number of peptide/HLA complex, flow cytometryusing the anti-AFP158/HLA-A*02:01 antibody was unable to detect asignificant MFI shift. In contrast, expression of an AFP minigene in theSK-HEP-1-AFP-MG cells resulted in one log shift in MFI by flowcytometry, indicating that the level of AFP158/HLA-A*02:01 complex onSK-HEP-1-AFP-MG cells was significantly higher than that in HepG2. WhenHepG2 was used as target cells and a panel of eight human cytokines(IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, IFN-γ, TNF-α) was measured after16 hours of co-incubation with abTCR- or CAR-transduced T cells, IL-8,IL-10, GM-CSF, IFN-γ, TNF-α was detectable in the media (FIG. 11A). Thecytokine release was consistently lower in the abTCR-transduced sampleswhen compared to the CAR-transduced samples. When the same assay wasdone with SK-HEP-1-AFP-MG, secretion of 7 out of the 8 cytokines that wetested were detected upon co-incubation with abTCR- or CAR-transducedprimary T cells (FIG. 11B). In the case of each cytokine tested, thelevel of cytokine detected in the media was either similar or lower insamples containing abTCR-transduced T cells compared with the CAR-Tsamples, some by more than two fold (e.g., IL-2, IFN-γ, TNF-α). SK-HEP-1cells alone also exhibited a detectable level (˜3000 pg/ml abovebackground) of IL-6 and IL-8 in the absence of T cells.

To determine the contribution of the transduced T cells as the source ofthe cytokines detected in the media, abTCR- and CAR-transduced T cellswith similar transduction efficiencies (34%) were co-cultured withtarget cells at a ratio of 2.5:1 with a protein transport inhibitorcocktail (eBioscience Cat #00498003) to prevent cytokine secretion.After 4 hours of treatment, T cells were stained with AFP158/HLA-A*02:01tetramer and anti-CD4 antibody along with anti-TNF-α, anti-IFN-γ,anti-IL-2 or anti-IL-6 antibodies. Using flow cytometry, gating onAFP158 tetramer⁺ cells (FIGS. 12A-12H), we demonstrated thatintracellular TNF-α, IFN-γ and IL-2, but not IL-6, were expressed inboth abTCR- and CAR-transduced T cells when they are co-cultured withantigen-positive target cells. For each cytokine examined, the level ofintracellular cytokine was consistently higher in SK-HEP-1-AFP-MG thanHepG2, correlating with the level of antigen expression on the targetcells. For each target cell population, there was no significantdifference in intracellular cytokines between abTCR- vs CAR-transducedcells, for each cytokine tested. This suggests that the difference seenin the cytokine release assay may be due to cytokine feedback. Theabsence of intracellular IL-6 in the transduced T cells suggests thatthe source of the IL-6 detected in the media in FIG. 11B is from theSK-HEP-1 cells and not the T cells.

To determine if the activation of abTCRs in CD4+ T cells could lead tospecific biological responses, we investigated the intracellularcytokine expression in CD4⁺, anti-AFP158 abTCR-expressing T cellsfollowing stimulation with cancer cell lines expressing AFP. CD3+ Tcells were transduced with the anti-AFP158 abTCR as described above andincubated with cancer cell line SK-HEP1-MG (AFP+), SK-HEP1 (AFP⁻), orHEPG2 (AFP⁺) for 4 hours in the presence of protein transporterinhibitor. As a negative control, abTCR-transduced T cells wereincubated in the absence of any cancer cell line. After the incubation,the T cells were stained with anti-IFNγ, anti-IL2, or anti-TNFαantibodies, and co-stained with AFP-tetramer-PE and anti-CD4. Cellsgated for abTCR expression were analyzed by flow cytometry forgranularity and cytokine expression (FIG. 13, Y-axis is side scattering,X-axis is cytokine staining). The expression of IFNγ, IL2, and TNFα wasinduced following incubation of the anti-AFP158 abTCR-transduced T cellswith AFP⁺ cancer cell lines SK-HEP1-MG and HEPG2, but not when incubatedwith AFP⁻ cell line SK-HEP1 or in the absence of any cancer cell line,indicating the antigen-specific activation of the abTCR in CD4+ T cells.

Expression of T Cell Exhaustion Markers in abTCR and CAR T Cells afterCo-Culture with Target Cells

To examine the level of exhaustion markers expressed on abTCR- andCAR-transduced cells upon antigen stimulation, CD3⁺ T cells wereprepared from PBMC-enriched whole blood using EasySep Human T CellIsolation Kit (StemCell Technologies) and activated with CD3/CD28Dynabeads as above. The activated and expanded cell population was >99%CD3+ by flow cytometry. These cells were then transduced with lentiviralvectors encoding a CAR containing an anti-AFP158/HLA-A*02:01 scFv (SEQID NO: 37) or an abTCR-6MD containing the same anti-AFP158/HLA-A*02:01variable domains (SEQ ID NOs: 35 and 36) for 7-9 days. The transducedcells were co-cultured with target cells for 16 hours at aneffector-to-target ratio of 2.5:1 and co-stained with AFP158 tetramerand anti-CD8 antibody, along with antibodies to exhaustion markers PD-1,TIM-3 or LAG-3. The level of exhaustion markers on the transduced Tcells were analyzed by flow cytometry by gating on the tetramer+ (i.e.,transduced) T cells. In an independent flow cytometry experiment, wedetermined that tetramer+ T cells that were CD8− were CD4+ (data notshown).

Upregulation of PD-1 was observed among CD8⁻tetramer⁺ T cells, whileupregulation of LAG-1 and TIM-3 were observed among CD8⁺tetramer⁺ Tcells, after exposure to target cells that express AFP (HepG2 andSK-HEP-1-AFP-MG) (FIG. 14). In all cases, the level of exhaustion markerupregulation observed on CAR-transduced T cells were equal or higherthan that observed on abTCR-transduced T cells. This suggests that abTCRtransduced T cells may cause lower level of T cell exhaustion, resultingin longer T cell persistances in vivo. The percent of cells positive foreach of the exhaustion markers in the tested conditions was determinedand is shown in Table 2.

TABLE 2 Target T cell PD1 (%) TIM3 (%) LAG3 (%) cell lines subset CARabTCR CAR abTCR CAR abTCR HEPG2 CD8 11 5.0 33 15 21 7.7 CD4 41 22 22 6.82.0 0.8 SK-HEP1 CD8 3.2 2.0 7.3 2.2 4.9 2.8 CD4 27 14 8.1 2.0 1.0 0.5SK-HEP1- CD8 42 35 45 34 88 81 AFP MG CD4 87 81 46 34 32 24 T cell onlyCD8 1.7 1.1 1.1 0.5 1.4 1.0 CD4 15 7.4 2.2 0.4 0.4 0.2Expression of T Cell Differentiation Markers in abTCR and CAR T Cells

To determine if anti-AFP158 abTCRs can delay the differentiation of Tcells during in vitro expansion, we measured the cell surface expressionof three T cell differentiation markers, memory T cell markers CCR7 andCD28, and terminal differentiation marker Granzyme B. T cells weretransduced with lentiviral vectors encoding a CAR containing ananti-AFP158/HLA-A*02:01 scFv (SEQ ID NO: 37) or an abTCR-6MD containingthe same anti-AFP158/HLA-A*02:01 variable domains (SEQ ID NOs: 35 and36), stained with antibodies against these markers, and analyzed by flowcytometry at day 10-12 after viral transduction (FIG. 15). The resultsshow that for both CD4⁺ and CD8⁺ T cells, abTCR T cells expressed moreCCR7 and CD28, but less Granzyme B, than CAR T cells, suggesting thatthe anti-AFP158 abTCR T cells were less differentiated than theanti-AFP158 CART cells after T cell expansion in vitro.

Comparison of Anti AFP abTCR-6MD and abTCR-7 Constructs

The cell growth of primary T cells transduced to express ananti-AFP158/HLA-A*02:01 abTCR-6MD was compared with that of T cellstransduced with an anti-AFP158/HLA-A*02:01 abTCR-7 having the sameantibody variable domains. 6.7×10⁵ T cells were activated by αCD³/αCD28beads (1:1 ratio) in the presence of 100 U/ml IL-2 on day 0. Activated Tcells were transduced with either a lentiviral vector encoding theanti-AFP158/HLA-A*02:01 abTCR-6MD (TCRδ chimera subunit having the aminoacid sequence of SEQ ID NO: 35 and TCRγ chimera subunit having the aminoacid sequence of SEQ ID NO: 36) or a lentiviral vector encoding theanti-AFP158/HLA-A*02:01 abTCR-7 (TCRδ chimera subunit having the aminoacid sequence of SEQ ID NO: 81 and TCRγ chimera subunit having the aminoacid sequence of SEQ ID NO: 82) at MOI 4 on day 1. Transduced T cellswere then cultured and expanded in the presence of IL-2 for 9-10 days.Cell numbers were counted on day 1, day 5, day 7, and day 9. As shown inFIG. 16A, the abTCR-6-MD transduced T cells grew faster than theabTCR-7-transduced T cells, with almost twice as many viable cells byday 9. Expression of the abTCR-6MD and abTCR-7 constructs in thetransduced T cells at day 9 was assessed by Western blot analysis forthe FLAG-tagged constructs. Briefly, 5 million transduced T cells werelysed in 100 μl lysis buffer and 13 μl of lysate was separated on a4-12% polyacrylamide gel using the NuPage system. Mouse anti-FLAGantibody (1 μg/ml) was used to detect abTCR gamma chains and mouseanti-CD3zeta (1 μg/ml) was used to detect endogenous CD3. As shown inFIG. 16B, the abTCR-7 construct was expressed at higher levels than theabTCR-6MD construct. The intensities of the anti-FLAG bands for thelysates, normalized to the corresponding anti-CD3ζ bands, werequantified using ImageJ software and showed a relative increase of 20%for the expression of the abTCR-7 compared to the abTCR-6MD.

The target-cell killing activity of the anti-AFP158/HLA-A*02:01abTCR-6MD T cells was compared with that of the anti-AFP158/HLA-A*02:01abTCR-7 T cells. Primary T cells were mock-transduced or transduced withlentiviral vectors encoding either the anti-AFP158/HLA-A*02:01 abTCR-6MDor anti-AFP158/HLA-A*02:01 abTCR-7 constructs described above. T cellstransduced with the abTCR-6MD or abTCR-7 were tested for their abilityto kill SK-HEP-1 (AFP−/HLA-A2+) and SK-HEP-1-AFP-MG (SK-HEP-1 transducedwith an AFP minigene) cells at an effector-to-target ratio of 5:1. Thelevel of specific killing was measured at 16 hours as described above.As shown in FIG. 16C, the abTCR-6MD construct with theanti-AFP158/HLA-A*02:01 binding moiety directed similar specific lysisof AFP-positive SK-HEP-1-AFP-MG cells as compared to the abTCR-7 withthe same antibody variable domains.

Example 5. Characterizing Biological Activities of T Cells Transducedwith abTCR-6MD and CAR Constructs Having the Same Anti-Human CD19Variable Domains

In example 4, the antibody moiety used was a TCR-mimic, which binds apeptide/MHC complex as antigen. To demonstrate that abTCR designs alsowork with traditional antibody targets (cell surface antigens), asimilar set of experiments was carried out using constructs based on anantibody against human CD19.

CD19 abTCR-Transduced T Cells can Kill CD19 Positive Cancer Cells

Primary T cells were mock-transduced or transduced with lentiviralvectors encoding a CAR containing an anti-CD19 binding domain (SEQ IDNO: 44, comprising a scFv with an IgV_(H) domain, SEQ ID NO: 45, and anIgV_(L) domain, SEQ ID NO: 46, from an exemplary anti-CD19 antibody) oran abTCR-6MD containing the same anti-CD19 variable domains (SEQ ID NOs:42 and 43). T cells transduced with CAR or abTCR (both at 25%transduction rate) were used to test their abilities to kill B celllines JeKo-1 (CD19⁺), IM9 (CD19⁺), Jurkat (CD19⁻), and THP-1 (CD19⁻), atan effector-to-target ratio of 5:1. The level of specific killing wasmeasured at 16 hours using the same method as described for FIG. 9B. Asshown in FIG. 17, both CAR and abTCR-6MD with an anti-CD19 bindingmoiety directed killing of CD19-positive JeKo-1 and IM9 cells at similarlevels, but did not kill Jurkat and THP-1, which are CD19-negative.

Cytokine Secretion by abTCR and CAR T Cells in Tumor Cell Killing

The same transduced T cell populations as used in the cancer killingexperiment above were used in a cytokine release assay by co-incubatingthem with JeKo-1, IM9, THP-1 and Jurkat as target cells. A panel ofeight human cytokines (IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, IFN-γ,TNF-α) was measured after 16 hours. All cytokines tested were detectedin the media of CAR-transduced T cells upon co-incubation with CD19⁺target cells, but not CD19⁻ cells (FIGS. 18A and 18B). In samplesco-incubated with abTCR-transduced T cells, the release for allcytokines tested, with the exception of IL-10, was lower in the abTCRsamples with CD19⁺ cells. These findings with the anti-CD19 antibodyconstructs are similar to those of the anti-AFP158/HLA-A*02:01 antibodyconstructs: while similar cancer cell killing was observed for CAR-T andabTCR-transduced cells, the level of cytokine release was lower whenabTCR-transduced T cells were used. This could be an advantage for usingabTCRs in settings where high levels of cytokine release causeundesirable physiological effects.

To determine if the activation of abTCRs in CD4+ T cells could lead tospecific biological responses, we investigated the intracellularcytokine expression in CD4⁺, anti-CD19 abTCR-expressing T cellsfollowing stimulation with cancer cell lines expressing CD19. CD3+ Tcells were transduced with Clone 5-13 abTCR-6MD (abTCR-6MD havinganti-CD19 clone 5-13 binding moiety which comprises amino acid sequencesof SEQ ID NOs: 56 and 54) and incubated with cancer cell line Raji(CD19⁺), Raji-CD19KO (CD19⁻), or Jeko-1 (CD19⁺) for 4 hours in thepresence of protein transporter inhibitor. As a negative control,abTCR-transduced T cells were incubated in the absence of any cancercell line. After the incubation, the T cells were stained withanti-IFNγ, anti-IL2, or anti-TNFα antibodies, and co-stained withanti-human Fab (CD19) and anti-CD4. Cells gated for abTCR expressionwere analyzed by flow cytometry for granularity and cytokine expression(FIGS. 19A-19C, Y-axis is side scattering, X-axis is cytokine staining).The expression of IFNγ, IL2, and TNFα was induced following incubationof the anti-CD19 abTCR-transduced T cells with CD19⁺ cancer cell linesRaji and Jeko-1, but not when incubated with CD19⁻ cell line Raji-CD19KOor in the absence of any cancer cell line, indicating theantigen-specific activation of the abTCR in CD4+ T cells.

Expression of T Cell Exhaustion Markers in abTCR and CAR T Cells afterCo-Culture with Target Cells

The level of exhaustion markers expressed on anti-CD19 abTCR- andCAR-transduced cells upon antigen stimulation was determined asdescribed above for anti-AFP158 chimeric receptors. The cells weretransduced with Clone 5-13 abTCR-6MD or a CAR containing the sameanti-CD19 variable domains. Target cell lines included Raji (CD19+),Raji-CD19KO (CD19−), and Jeko-1 (CD19+). The percent of cells positivefor each of the exhaustion markers in the tested conditions wasdetermined and is shown in Table 3.

TABLE 3 Target T cell PD1 (%) TIM3 (%) LAG3 (%) cell lines subset CARabTCR CAR abTCR CAR abTCR Raji CD8 14 4.0 47 37 95 93 CD4 74 41 29 24 6547 Raji- CD8 2.9 0.3 54 35 40 29 CD19KO CD4 27 7.0 12 13 5.3 4.1 Jeko-1CD8 14 4.7 48 40 92 76 CD4 70 33 36 30 58 31 T cell only CD8 1.7 0.2 5.19.8 9.4 5.6 CD4 15 2.2 0.3 1.0 1.1 1.0Expression of T Cell Differentiation Markers in abTCR and CAR T Cells

To determine if anti-CD19 abTCRs can delay the differentiation of Tcells during in vitro expansion, we measured the cell surface expressionof three T cell differentiation markers, memory T cell markers CCR7 andCD28, and terminal differentiation marker Granzyme B. T cells weretransduced with Clone 5-13 abTCR-6MD or a CAR having the same anti-CD19variable domains, stained with antibodies against these markers, andanalyzed by flow cytometry at day 10-12 after viral transduction (FIG.20). The results show that for both CD4⁺ and CD8⁺ T cells, the abTCR Tcells expressed more CCR7 and CD28, but less Granzyme B, than the CAR Tcells, suggesting that the Clone 5-13 abTCR T cells were lessdifferentiated than the corresponding CAR T cells after T cell expansionin vitro, in agreement with what was observed for anti-AFP158 chimericreceptors.

Proliferation of abTCR and CAR T Cells

To further determine if abTCR T cells are less differentiated and havehigher proliferation potential than CAR T cells, we monitored the changein CFSE fluorescence, an indicator of cell division, of abTCR and CAR Tcells after their engagement with antigen-positive cancer cells. The Tcells were labeled with CFSE dye at day 10 after viral transduction withClone 5-13 abTCR-6MD or a CAR having the same anti-CD19 variabledomains, and baseline fluorescence was recorded by flow cytometry. Thelabeled T cells were incubated with Raji cells (a CD19+ cancer cellline) in cytokine-free medium. The CFSE fluorescence was measured byflow cytometry at day 2 and day 3 for CD4+ and CD8+ T cells (FIG. 21).The decrease in CFSE fluorescence intensity between day 2 and day 3,indicating amount of cell proliferation, was significantly higher inabTCR T cells than CAR T cells, indicating that the Clone 5-13 abTCR Tcells undergo more cell divisions than the corresponding CAR T cells.

Chimeric Receptor Internalization of abTCR and CAR T Cells

To compare the internalization rate of T cell surface abTCRs and CARs, Tcells were transduced with Clone 5-13 abTCR-6MD or a CAR containing thesame anti-CD19 variable domains, and stained on ice for 30 minutes withan anti-idiotype antibody recognizing the anti-CD19 binding moietylabeled with CypHerSE, a PH sensitive dye that emits fluorescence atacidic pH 6.5. The cells were then incubated at 37° C. for the indicatedamount of time, fixed, and analyzed by flow cytometry for granularityand chimeric receptor expression (FIG. 22, Y-axis is side scattering,X-axis is CypHerSE staining). The results show that almost all the CARwas internalized by 90 minutes after staining. In contrast, the abTCRwas internalized at a much slower rate, and most of the abTCR remainedon the cell surface even at 90 minutes.

Comparison of Anti-CD19 abTCR and cTCR Constructs

The cell growth of primary T cells transduced to express an anti-CD19abTCR-6MD was compared with that of T cells transduced with an anti-CD19chimeric construct (cTCR) having the same antibody variable domains andtransmembrane domains but with constant regions from TCRδ and TCRγpolypeptides. 6.7×10⁵ T cells were activated by αCD³/αCD28 beads (1:1ratio) in the presence of 100 U/ml IL-2 on day 0. Activated T cells weretransduced with either a lentiviral vector encoding the anti-CD19abTCR-6MD (TCRδ chimera subunit having the amino acid sequence of SEQ IDNO: 56 and TCRγ chimera subunit having the amino acid sequence of SEQ IDNO: 54) or a lentiviral vector encoding the anti-CD19 cTCR (TCRδ chimerasubunit having the amino acid sequence of SEQ ID NO: 75 and TCRγ chimerasubunit having the amino acid sequence of SEQ ID NO: 76) at MOI 4 onday 1. Transduced T cells were then cultured and expanded in thepresence of IL-2 for 9-10 days. Cell numbers were counted on day 1, day5, day 7, and day 9. As shown in FIG. 23A, the abTCR-transduced T cellsgrew faster than the cTCR-transduced T cells, with more than 1.7 timesas many viable cells counted at day 9.

The target-cell killing activity of the anti-CD19 abTCR T cells wascompared to that of the anti-CD19 cTCR T cells. Primary T cells weremock-transduced or transduced with lentiviral vectors encoding eitherthe anti-CD19 abTCR-6MD or the anti-CD19 cTCR. T cells transduced withthe abTCR or cTCR were tested for their ability to kill CD19-positivetarget cell line Nalm-6 at an effector-to-target ratio of 5:1. The levelof specific killing was measured at 16 hours as described above. Asshown in FIG. 23B the abTCR-6MD construct with the anti-CD19 bindingmoiety directed greater specific lysis of CD19-positive Nalm-6 cellsthan the cTCR with the same anti-CD19 variable domains.

Example 6. Characterizing Biological Activities of T Cells Transducedwith abTCR-6MD and CAR Constructs Having the SameAnti-NY-ESO-1/HLA-A*02:01 Variable Domains

Anti-NY-ESO-1/HLA-A*02:01 abTCR-Transduced T Cells can KillNY-ESO-1-Positive Cancer Cells

Primary T cells were mock-transduced or transduced to express either aCAR or abTCR-6MD containing an anti-NY-ESO-1/HLA-A*02:01 binding moietycomprising an IgV_(L) domain having the amino acid sequence of SEQ IDNO: 73 and an IgV_(H) domain having the amino acid sequence of SEQ IDNO: 72. The CAR comprised an scFv having, from N-terminus to C-terminus,the IgV_(L) domain, a linker (SEQ ID NO: 74), and the IgV_(H) domain. Tcells transduced with the CAR or abTCR expressed their respectivechimeric receptor at similar levels as assayed by flow cytometry andwere used to test their abilities to kill cell lines IM9 (HLA-A2⁺,NY-ESO-1⁺), Colo205 (HLA-A2⁺, NY-ESO-1⁻), MDA-231 (HLA-A2⁺, NY-ESO-1⁻),MCF7 (HLA-A2⁺, NY-ESO-1⁻), JeKo-1 (HLA-A2⁺, NY-ESO-1⁺), Raji (HLA-A2⁺,NY-ESO-1⁻), Hep1 (HLA-A2⁺, NY-ESO-1⁻), and Jurkat (HLA-A2⁺, NY-ESO-1⁻)at an effector-to-target ratio of 5:1. The level of specific killing wasmeasured at 16 hours using the same methods described above. As shown inFIG. 24, both the CAR and abTCR-6MD with the anti-NY-ESO-1/HLA-A*02:01binding moiety directed killing of NY-ESO-1-positive JeKo-1 and IM9cells at similar levels, but did not kill the other cells, which areNY-ESO-1-negative.

Cytokine Secretion by abTCR and CAR T Cells in Tumor Cell Killing

The same transduced T cell populations as used in the cancer killingexperiment above were used in a cytokine release assay by co-incubatingthem with IM9, Colo205, MDA-231, MCF7, JeKo-1, Hep1, and Jurkat cells. Apanel of four human cytokines (IL-2, GM-CSF, IFN-γ, TNF-α) was measuredafter 16 hours. All cytokines tested were detected in the media of CAR-and abTCR-transduced T cells upon co-incubation with NY-ESO-1⁺ targetcells, but not most NY-ESO-1⁻ cells (data not shown). Importantly, thelevels of cytokines released from most of the tested NY-ESO-1⁺ targetcells by abTCR-transduced T cell co-incubation were significantly lowerthan by CAR-transduced T cell co-incubation (data not shown).

Example 7. Characterizing Biological Activities of Natural Killer T(NKT) Cells and Regulatory T (Treg) Cells Transduced with abTCR-6MDConstructs

Anti-CD19 abTCR-Transduced NKT Cells

NKT cells were isolated from human PBMCs by indirect magnetic labellingof non-CD3⁺/CD56⁺ cells (non-NKT cells) with a biotin-antibody cocktailand anti-biotin microbeads, and depleting the non-NKT cells to enrichfor CD3⁺/CD56⁺ NKT cells. The surface expression of CD3 and CD56 for theenriched NKT cell population was assessed by flow cytometry and is shownin FIG. 25A. The NKT cells were activated by anti-CD³/anti-CD28 beads,transduced with lentivirus encoding the anti-CD19 abTCR, and expanded inRPMI-1640 containing 10% FBS and IL-2 (100 U/ml). Transductionefficiency was greater than 80% as measured by flow cytometry with ananti-idiotype antibody specific for the anti-CD19 binding moiety. TheNKT cells were co-incubated with CD19-expressing Raji or RajiCD19-knockout (CD19ko) cancer cell lines at an effector-to-target ratioof 5:1 for 16 hours followed by measurement of cytokine release (IL-2,GM-CSF, IFNγ, TNFα) in the media (FIG. 25B). The anti-CD19abTCR-transduced NKT cells, but not mock-transduced NKT cells, wereactivated to release each of the cytokines tested when incubated withthe CD19− positive Raji cells, but not the CD19-negative Raji CD19kocells, indicating that NKT cells can be specifically activated throughbinding of the transduced abTCR with the CD19 antigen on cancer cells.

Anti-CD19 abTCR-Transduced Treg Cells

Treg cells were isolated from human PBMCs by direct magnetic labellingof CD4⁺/CD25⁺ Treg cells. The surface expression of CD4 and CD25 for theisolated Treg cell population was assessed by flow cytometry and isshown in FIG. 26A. The Treg cells were activated by anti-CD³/anti-CD28beads, transduced with lentivirus encoding the anti-CD19 abTCR, andexpanded in RPMI-1640 containing 10% FBS and IL-2 (100 U/ml).Transduction efficiency was 80% as measured by flow cytometry with ananti-idiotype antibody specific for the anti-CD19 binding moiety. TheTreg cells were co-incubated with CD19-expressing Raji or RajiCD19-knockout (CD19ko) cancer cell lines at an effector-to-target ratioof 5:1 for 16 hours followed by measurement of IL-10 cytokine release inthe media (FIG. 26B). The anti-CD19 abTCR-transduced Treg cells, but notmock-transduced Treg cells, were activated to release IL-10 whenincubated with the CD19-positive Raji cells, but not the CD19-negativeRaji CD19ko cells, indicating that Treg cells can be specificallyactivated through binding of the transduced abTCR with the CD19 antigenon cancer cells.

Example 8. Characterizing Biological Activities of T Cells Transducedwith abTCRs Having Different Antibody Heavy Chain Constant Domains

In the previous examples, the antibody moieties used in the abTCRconstructs contained an IgG1 CH1 domain having the amino acid sequenceof SEQ ID NO: 39. To demonstrate that abTCR designs also work with CH1domains from other immunoglobulin heavy chains, target-cell killingassays were carried out as described above using constructs based on ananti-AFP158/HLA-A*02:01 antibody having CH1 domains from either IgG1(SEQ ID NO: 39), IgG2 (SEQ ID NO: 60, 61, or 62), IgG3 (SEQ ID NO: 63),or IgG4 (SEQ ID NO: 64). T cells transduced with the abTCRs were assayedfor AFP158 tetramer binding as an indication of surface expression(Table 4) and tested for their ability to kill HepG2 (AFP+/HLA-A2+),SK-HEP-1 (AFP−/HLA-A2+), and SK-HEP-1-AFP-MG (SK-HEP-1 transduced withan AFP minigene) cells. Specific lysis was measured after 16 hrincubation using the Cytox 96 Non-radioactive Cytotoxicity Assay(Promega). As shown in FIG. 27, T cells transduced with any of theabTCRs bearing the anti-AFP158/HLA-A*02:01 binding moiety directedkilling of antigen-positive cell lines HepG2 and SK-HEP-1-AFP-MG, butdid not lead to killing of antigen-negative cell line SK-HEP-1.Importantly, even though surface expression of the abTCRs containingnon-IgG1 CH1 domains was lower compared to the abTCR containing IgG1 CH1(see Table 4), they resulted in similar levels of target cell killing,suggesting they may have enhanced functional properties.

TABLE 4 abTCR surface expression AFP158 tetramer+ abTCR (percent) Mock0.3 IgG1 64.5 IgG2-0C 9.58 IgG2-1C 18.2 IgG2-2C 7.36 IgG3 13.6 IgG4 22.2

Example 9. Characterizing Biological Activities of T Cells Transducedwith abTCRs Containing Co-Stimulatory Domains

To demonstrate the feasibility of abTCR designs including C-terminalco-stimulatory domains, various anti-AFP158/HLA-A*02:01 abTCR constructswere designed using co-stimulatory fragments derived from CD28 and/or4-1BB (FIG. 28). The abTCRs consisted of a TCRγ chimeric subunitcontaining the amino acid sequence of SEQ ID NO: 36 and a TCRδ chimericsubunit containing the amino acid sequence of SEQ ID NO: 35. The abTCRsubunits having a CD28 co-stimulatory domain had the amino acid sequenceof SEQ ID NO: 70 fused to their C-terminus, and subunits having a 4-1BBco-stimulatory domain had the amino acid sequence of SEQ ID NO: 71 fusedto their C-terminus. The abTCR constructs were transduced intoJ.RT3-R3.5 cells, and abTCR expression and CD3 surface expression rescuewas assayed as described above using flow cytometry. The results aresummarized in Table 5. The expression of the abTCRs and their ability torescue CD3 expression was similar between the various abTCR constructswith and without co-stimulatory domains. In another experiment, primaryT cells were transduced with the abTCR constructs and assayed by flowcytometry for CD8 expression and AFP158 tetramer binding (Table 6).Primary T cells transduced with the abTCRs were gated for AFP158tetramer binding and either CD4 or CD8 expression and assayed by flowcytometry for expression of CCR7, CD45RA, CD28, and Granzyme B (Table7). These results show that viral transduction and differentiation oftransduced T cells was similar between the various abTCR constructs withand without co-stimulatory domains. Target-cell killing assays werecarried out as described above using the abTCR constructs. T cellstransduced with the abTCRs were assayed for their ability to kill HepG2(AFP+/HLA-A2+), SK-HEP-1 (AFP−/HLA-A2+), and SK-HEP-1-AFP-MG (SK-HEP-1transduced with an AFP minigene) cells. As shown in FIG. 29, T cellstransduced with any of the abTCRs directed killing of antigen-positivecell lines HepG2 and SK-HEP-1-AFP-MG, but did not lead to killing ofantigen-negative cell line SK-HEP-1.

To further characterize the abTCR constructs containing co-stimulatorydomains, T cells transduced with the various abTCRs were gated into 4different populations (CD8+/abTCR+; CD8+/abTCR−; CD4+/abTCR+;CD4+/abTCR−) and assayed by flow cytometry for expression of the T cellexhaustion markers PD-1, TIM-3, and LAG-3 following incubation withHepG2, SK-HEP-1, and SK-HEP-1-AFP-MG cells. T cells transduced with thevarious abTCRs containing C-terminal co-stimulatory domains did not showsignificantly increased T cell exhaustion following activation by thetarget-positive cells HepG2 and SK-HEP-1-AFP-MG as compared to the Tcells transduced with the abTCR lacking any co-stimulatory domains (datanot shown). T cells transduced with the various abTCRs were used in acytokine release assay as described above by co-incubating them withSK-HEP-1 and SK-HEP-1-AFP-MG cells. A panel of four human cytokines(IL-2, GM-CSF, IFN-γ, TNF-α) was measured after 16 hours. All cytokinestested were detected in the media of abTCR-transduced T cells uponco-incubation with AFP+SK-HEP-1-AFP-MG cells, but not AFP⁻ SK-HEP-1cells (FIG. 30). T cells transduced with the various abTCRs containingC-terminal co-stimulatory domains did not show significantly increasedcytokine release following activation by the target-positiveSK-HEP-1-AFP-MG cells as compared to the T cells transduced with theabTCR lacking any co-stimulatory domains, and in some cases showedreduced cytokine release.

The target-cell killing, T cell exhaustion, and cytokine releaseexperiments were repeated using anti-CD19 abTCR constructs designedusing co-stimulatory fragments derived from CD28 and/or 4-1BB asdescribed above and depicted in FIG. 28. The abTCRs consisted of a TCRγchimeric subunit containing the amino acid sequence of SEQ ID NO: 54 anda TCRδ chimeric subunit containing the amino acid sequence of SEQ ID NO:56. As with the anti-AFP158/HLA-A*02:01 abTCR constructs, the T cellstransduced with the anti-CD19 abTCRs containing co-stimulatory domainsbehaved similarly to T cells transduced with the anti-CD19 abTCRconstruct without any co-stimulatory domains (FIGS. 31 and 32).

TABLE 5 abTCR and CD3 surface expression abTCR⁺/CD3⁻ abTCR⁻/CD3⁺abTCR⁺/CD3⁺ abTCR (percent) (percent) (percent) Mock 0.02 2.49 0.03abTCR-6M 1.11 0.217 93.8 abTCR-6M-1 1.11 0.217 93.8 abTCR-6M-2 1.310.259 93.5 abTCR-6M-3 2.8 0.418 83.5 abTCR-6M-4 4.43 0.682 77.6abTCR-6M-5 3.51 0.685 81.3 abTCR-6M-6 2.91 0.738 77.3 abTCR-6M-7 2.81.17 66.4 abTCR-6M-8 3.68 0.892 75.5

TABLE 6 CD8 and abTCR surface expression abTCR CD8⁺/tetramer⁺CD8⁻/tetramer⁺ Mock 0.068 0.013 abTCR-6M 20.6 38.3 abTCR-6M-1 28.0 44.7abTCR-6M-2 23.1 39.2 abTCR-6M-3 24.9 40.0 abTCR-6M-4 18.2 37.1abTCR-6M-5 14.3 33.3 abTCR-6M-6 16.1 32.6 abTCR-6M-7 19.6 38.8abTCR-6M-8 7.79 19.6

TABLE 7 CCR7, CD45RA, CD28, and Granzyme B surface expression on CD4+and CD8+ abTCR T cells Percent Expression Percent Expression(CD4⁺/tetramer⁺-gated) (CD8⁺/tetramer⁺-gated) Granzyme Granzyme abTCRCD28 CCR7 B CD28 CCR7 B abTCR-6M 60 56 2.4 52 26.5 23.3 abTCR-6M-1 65.953.7 2.31 57.1 20.9 21.6 abTCR-6M-2 60.3 53.8 2.41 57.1 19.1 28.5abTCR-6M-3 60.1 53.7 2.96 54.6 18.9 29.1 abTCR-6M-4 63 52.5 3.11 58.620.2 30.1 abTCR-6M-5 56.1 54 3.74 52.2 19.1 34.1 abTCR-6M-6 62.3 54.83.1 54.5 19 33.4 abTCR-6M-7 63 52.2 2.4 57.7 18.4 28.1 abTCR-6M-8 55.654.1 2.44 57.7 22.8 34.2

Example 10. In Vivo Efficacy Studies of abTCR-Transduced T Cells

In Vivo Antitumor Activity for Anti-AFP158/HLA-A*02:01 Antibody in aHuman Hepatocellular Carcinoma Xenograft Model

The in vivo antitumor activity of T cells transduced with abTCR-6MDconstructs containing an anti-AFP158/HLA-A*02:01 binding moiety (SEQ IDNOs: 35 and 36) was tested using a subcutaneous (s.c.) model ofSK-HEP-1-AFP-MG in SCID-beige mice. The SK-HEP-1-AFP-MG cells were s.c.implanted over the right flank of the SCID-beige mice at 5×10⁶ cells permouse. When average tumor volume reached 100 mm³, animals wererandomized based on tumor volume to two groups (with 8 mice per group)receiving: (i) mock-transduced T cells and (ii) abTCR-transduced Tcells. The animals were treated immediately after randomization byinjecting 10⁷ mock or abTCR-transduced per mouse, intravenously (i.v.)once every two weeks, for three doses. The mice were closely monitoredfor general health condition, possible adverse response, if any, andchanges in tumor volume. Both mock and the abTCR-transduced T cells werewell-tolerated at the current dose and schedule. No dosing-related bodyweight change was observed throughout the study (FIG. 33). WhileSK-HEP-1-AFP-MG tumors continued to grow after i.v. administration ofmock or abTCR-transduced T cells, the growth rate of abTCR-transduced Tcell treated tumors was slower compared to mock T-treated tumors. Asshown in FIG. 34A, the separation of the tumor growth curves started at20 days post dosing initiation. On day 31, 23% growth inhibition inabTCR-transduced T cell treated tumors were observed (t test, p=0.018).

The antitumor activity of abTCR-transduced T cells was further evaluatedin larger SK-HEP-1-AFP-MG s.c. tumors. In a study with SK-HEP-1-AFP-MGtumor-bearing mice, animals were randomized into two groups when averagetumor volume reached 300 mm³ (n=4 mice per group). Animals receivedeither no treatment or a single intratumoral (i.t.) injection of 10⁷abTCR-transduced T cells per mouse. As shown in FIG. 34B, i.t. deliveryof abTCR-transduced T cells slowed down the growth of largeSK-HEP-1-AFP-MG tumors as measured by change in tumor volume over time.Comparison of the areas under the curve between untreated andabTCR-transduced T cell-treated large SK-HEP-1-AFP-MG tumors showed astatistically significant difference between the two groups (two tailedt test, p=0.04). Taken together, both i.v. and i.t. administration ofabTCR-transduced T cells significantly inhibited the growth ofestablished s.c. xenografts of SK-HEP-1-AFP-MG.

In Vivo Antitumor Activity for Anti-CD19 Antibody in a LymphomaXenograft Model

The in vivo antitumor activity of T cells transduced with CAR and abTCRwith an exemplary anti-hCD19 antibody binding moiety are tested in CD19positive human lymphoma xenograft model in NOD SCID gamma (NSG) mice.Raji-luc-GFP cells are purchased from Comparative Biosciences, Inc.(Sunnyville, Calif. 94085) and are cultured in RPMI Medium+10% FBS and1% L-Glutamine at 37° C. in a humidified atmosphere with 5% CO2. TheRaji-luc-GFP cells are derived from the CD19-positive Burkitt lymphomacell line, Raji, after stable transfection with dual reporter genesencoding both firefly luciferase (luc) and green fluorescent protein,resulting in cells that can be traced in vivo using bioluminescentimaging. NSG mice are purchased from Jackson Laboratories (Bar Harbor,Me. USA 04609) and are acclimated for at least 7 days prior to theexperiment. Raji-luc-GFP cells are re-suspended in PBS and implantedintravenously (i.v.) into NSG mice through tail vein at 1×10⁶ cells/100μl/mouse. Five days post tumor implantation, animals are imaged usingXenogen IVIS imaging system for assessment of tumor burden. Mice arerandomized based on the photon emission into the following four groupsat average photon emission of 6.7×10⁵ photons (n=6 mice per group): (i)no treatment, (ii) mock-transduced human T cells, (iii) anti-CD19 CAR-Ttreated and (iv) anti-hCD19 abTCR T cells treated. The animals aretreated i.v. with mock or anti-CD19 CAR-T cells immediately afterrandomization at a dose of 10⁷ cells per mouse, once every two weeks for3 doses.

Animals are closely monitored after dosing. Bioluminescent imaging usingXenogen IVIS system is taken once a week for up to 8 weeks.

Animal studies were carried out as described above to evaluate in vivoanti-tumor capabilities of T cells transduced with abTCR-6MD havinganti-CD19 binding moieties.

6-8 weeks old female NSG mice were used in this study. The Raji-luc-GFPcell line was cultured in RPMI Medium+10% FBS and 1% L-Glutamine at 37°C. in a humidified atmosphere with 5% CO2. Raji-luc-GFP cells werere-suspended in PBS and implanted i.v. into 40 NSG mice at 1×10⁶cells/100 μl/mouse.

At four days post tumor implantation, the mice were imaged using theIvis Spectrum to confirm tumor growth. The mice were then randomized,based on the photon emission, into six groups for the followingtreatments (n=6 mice/group): 1) Vehicle (PBS); 2) Mock (8×10⁶mock-transduced T cells); 3) Clone 5 abTCR (8×10⁶ T cells transducedwith an abTCR-6MD having anti-CD19 clone 5 binding moiety whichcomprises amino acid sequences of SEQ ID NOs: 42 and 54); 4) Clone 5-3abTCR (8×10⁶ T cells transduced with an abTCR-6MD having anti-CD19 clone5-3 binding moiety which comprises amino acid sequences of SEQ ID NOs:42 and 43); 5) Clone 5-9 abTCR (8×10⁶ T cells transduced with anabTCR-6MD having anti-CD19 clone 5-9 binding moiety which comprisesamino acid sequences of SEQ ID NOs: 55 and 54); and 6) Clone 5-13 abTCR(8×10⁶ T cells transduced with an abTCR-6MD having anti-CD19 clone 5-13binding moiety which comprises amino acid sequences of SEQ ID NOs: 56and 54).

Animals were closely monitored after tumor implantation and dosing with8 million receptor-positive T cells. Animals were weighed and Xenogenimaging was conducted twice a week for the duration of the study.Animals showing the following conditions were euthanized and recorded as“conditional death”: a) acute adverse response: labored breathing,tremor, passive behavior (loss of appetite and lethargy); b) body weightloss more than 25% initial body weight; and c) limb paralysis thataffect mouse movement.

The results of this experiment are depicted in FIG. 35, which plotstotal flux emission from the tumor vs. days post dosing with abTCR cellsor controls. All 4 of the CD19-abTCR T cells targeted and lysed the CD19positive Raji tumors in vivo, demonstrating efficacy of anti-CD19antibodies in the abTCR platform to inhibit tumor growth.

In another experiment, NSG mice with no implanted tumors were treatedwith 8×10⁶ T cells transduced with an anti-CD19 abTCR-6MD or ananti-CD19 CAR with the same binding sequences, and the effect of thesetransduced T cells in vivo were compared. The mice treated with theanti-CD19 CAR T cells died within 24 hours, while the mice treated withthe anti-CD19 abTCR T cells survived after 5 weeks. This resultindicates that T cells expressing abTCR constructs are safer than thoseexpressing CARs.

Comparison of Anti-CD19 abTCR and Anti-CD19 CAR

Raji B-cell lymphoma Raji-luc-GFP cells were implanted into NSG mice asdescribed above. Mice were then injected with 5×10⁶ abTCR⁺ T cellstransduced with Clone 5-13 abTCR-6MD (abTCR-6MD having anti-CD19 clone5-13 binding moiety which comprises amino acid sequences of SEQ ID NOs:56 and 54), 5×10⁶ CAR⁺ T cells transduced with Clone 5-13 CAR (CARhaving anti-CD19 clone 5-13 binding moiety), or 5×10⁶ mock T cells ingroups of eight mice per injection sample. Serum was collected 24 hoursafter T cell implantation and the concentration of human cytokineswithin the serum was measured using the Luminex Magpix machine asdescribed above. The cytokine measurement results are shown in FIG. 36.Tumor burden was measured by luciferase activity as previously describedand results are shown in FIGS. 37 (quantitation) and 27 (imaging).

In a head to head comparison between Clone 5-13 CAR and Clone 5-13 abTCRT cells, abTCR T cells injected into mice resulted in rapid tumorregression compared to CAR T cells at early time points, while miceinjected with CAR T cells did not show tumor regression until afterabout five days. Throughout the course of this experiment, Clone 5-13abTCR T cells showed higher in vivo tumor inhibition efficacy than Clone5-13 CAR T cells. The cytokine measurement results at 24 hrs indicatethat mice treated with Clone 5-13 abTCR T cells also had reducedcytokine secretion levels than CAR T cell-treated mice. These resultsprovide evidence that anti-tumor efficacy does not necessitateover-production of cytokines, as abTCR T cells have higher tumorinhibition potency yet lower cytokine-secreting effects than CAR Tcells.

At 7 weeks following tumor implantation, none of the mice treated withClone 5-13 abTCR T cells had detectable tumors. At this time, 3 micefrom the mock T cell treated group and 3 mice from the Clone 5-13 abTCRT cell treated group were re-challenged by i.v. implantation with 5×10⁵Raji lymphoma cells. Tumor burden was measured by luciferase activity aspreviously described and results are shown in FIG. 39. While tumors grewrapidly in mice from the mock T cell treated group, the prior treatmentwith Clone 5-13 abTCR-transduced T cells prevented the growth of tumorsfollowing re-challenge with Raji lymphoma cell implantation, indicatingthat the abTCR-transduced T cells persisted and maintained their abilityto respond to antigen.

In another experiment, serum was collected from NSG mice 24 hours afterinjection of either 5×10⁶ Clone 5-3 abTCR-6MD (abTCR-6MD havinganti-CD19 clone 5-3 binding moiety which comprises amino acid sequencesof SEQ ID NOs: 42 and 43) T cells or 5×10⁶ Clone 5-3 CAR (CAR havinganti-CD19 clone 5-3 binding moiety, SEQ ID NO: 44) T cells. Theconcentration of cytokines in the mouse serum was measured as describedabove. High levels of T cell-derived human cytokines and mouse-derivedIL-6 were found in mice treated with Clone 5-3 CAR T cells. In contrast,mice treated with Clone 5-3 abTCR displayed dramatically lower serumcytokine levels (data not shown), providing further evidence of thereduced effect on cytokine overproduction for abTCR T cells.

In Vivo Antitumor Activity for Anti-CD19 Antibody in a LeukemiaXenograft Model

The in vivo antitumor activity of T cells transduced with a CAR or abTCRcontaining an exemplary anti-hCD19 antibody binding moiety were testedin a CD19 positive human leukemia xenograft model in NSG mice.NALM-6-luc-GFP cells were a gift from Eric Smith's Lab at Memorial SloanKettering Cancer Center and were cultured in RPMI Medium+10% FBS at 37°C. in a humidified atmosphere with 5% CO2. NALM-6-luc-GFP cells arederived from the CD19-positive acute lymphoblastic leukemia cell line,NALM-6, after stable transfection with dual reporter genes encoding bothfirefly luciferase (luc) and green fluorescent protein, resulting incells that can be traced in vivo using bioluminescent imaging. NSG micewere purchased from Jackson Laboratories (Bar Harbor, Me. USA 04609) andacclimated for at least 3 days prior to the experiment. NALM-6-luc-GFPcells were re-suspended in PBS and implanted intravenously (i.v.) intothirty 6-8 week-old female NSG mice through tail vein at 5×10⁵ cells/100μl/mouse. Four days post tumor implantation, animals were imaged usingXenogen IVIS imaging system for assessment of tumor burden. Mice wererandomized based on the photon emission into the following four groups:(i) Vehicle, PBS only (n=6 mice); (ii) 10×10⁶ mock-transduced human Tcells (n=6 mice); (iii) 5×10⁶ Clone 5-13 CART cells (T cells transducedwith a CAR having anti-CD19 clone 5-13 binding moiety) (n=8 mice); and(iv) 5×10⁶ Clone 5-13 abTCR-6MD T cells (T cells transduced with anabTCR-6MD having anti-CD19 clone 5-13 binding moiety which comprisesamino acid sequences of SEQ ID NOs: 56 and 54) (n=8 mice).

Animals were closely monitored after tumor implantation and dosing withreceptor-positive T cells. Animals were weighed and Xenogen imaging wasconducted twice a week for the duration of the study. Animals showingthe following conditions were euthanized and recorded as “conditionaldeath”: a) acute adverse response: labored breathing, tremor, passivebehavior (loss of appetite and lethargy); b) body weight loss more than25% initial body weight; and c) limb paralysis that affect mousemovement.

The results of this experiment are depicted in FIG. 40, which plots theaverage tumor-derived total flux emissions for each treatment arm vs.days post treatment. Both the Clone 5-13 abTCR T cells and Clone 5-13CAR T cells targeted and lysed the CD19 positive NALM-6 tumors in vivo,demonstrating the efficacy of anti-CD19 antibodies in the abTCR platformto inhibit tumor growth in multiple cancer models.

At 24-hours post treatment, blood was collected from 3 mice per groupfor cytokine measurements, and the results are shown in FIG. 41. As withthe lymphoma xenograft model, treatment with anti-CD19 abTCR T cells inthis leukemia xenograft model resulted in lower levels of cytokinesecretion than treatment with anti-CD19 CART cells.

At 7 days and 13 days post treatment, blood was collected fromrepresentative mice from each group and analyzed by flow cytometry usingthe “123count eBeads” kit from Affymetrix eBioscience, Inc. to determinethe numbers of CD3+ T cells, CAR/abTCR-expressing T cells, and tumorcells per μl of blood, and the level of PD-1 expression on T cells. At13 days post treatment, 2 mice per group were euthanized and bone marrowextracts were analyzed by flow cytometry for CD3+/CAR/abTCR T cells, thepresence of tumor cells, and PD-1 expression levels on T cells.

Mice administered abTCR T cells had higher levels of chimericreceptor-expressing T cells in their blood at both day 7 and 13following administration than was observed for mice administered CAR Tcells (FIG. 42), indicating that abTCR T cells had higher levels ofviability and/or proliferation than their counterpart CAR T cells inthis model. As shown in FIGS. 43 and 44, while mice treated with eitherCAR T cells or abTCR T cells showed a reduction in tumor cells(indicated by FITC staining) in both peripheral blood and bone marrowcompared to vehicle- and mock-treated control animals at 13 days posttreatment, the reduction in tumor cells in both peripheral blood andbone marrow was greater for animals treated with abTCR T cells. As shownin FIGS. 45 and 46, the expression level of PD-1, a T cell exhaustionmarker, on the surface of T cells from both peripheral blood and bonemarrow was lower in mice treated with abTCR T cells than those treatedwith CAR T cells, and comparable to levels observed in mock-treatedmice, for both CD4⁺ and CD8⁺ T cells. These results suggest thatabTCR-expressing T cells may be less likely to become exhausted thanCAR-expressing T cells.

Sequence Listing SEQ ID NO Description Sequence 1 TCRα transmembraneILLLKVAGFNLLMTLRLWSS domain 2 TCRβ transmembrane TILYEILLGKATLYAVLVSALVLdomain 3 TCRδ transmembrane MLFAKTVAVNFLLTAKLFFL domain 4TCRγ transmembrane YYMYLLLLLKSVVYFAIITCCLL domain 5 TCRα connectingESSCDVKLVEKSFETDTNLNFQNLSVIGFR peptide 6 TCRβ connectingADCGFTSVSYQQGVLSA peptide 7 TCRδ connectingDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLR peptide 8 TCRγ connectingMDPKDNCSKDANDTLLLQLTNTSA peptide 9 TCRα connectingIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFR peptide MD 10 TCRβ connectingGRADCGFTSVSYQQGVLSA peptide MD 11 TCRδ connectingEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLG peptide MD LR 12TCRγ connecting PIKTDVITMDPKDNCSKDANDTLLLQLTNTSA peptide MD 13TCRβ intracellular MAMVKRKDF domain 14 TCRγ intracellular RRTAFCCNGEKSdomain 15 TCRD alpha ESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRL WSS16 TCRD beta ADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKR KDF 17TCRD alpha MD IPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 18 TCRD beta MDGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV KRKDF 19 TCRD deltaDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFA KTVAVNFLLTAKLFFL 20TCRD gamma MDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCL LRRTAFCCNGEKS21 TCRD delta MD EVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 22 TCRD gamma MDPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVY FAIITCCLLRRTAFCCNGEKS 23anti-AFP158/HLA- EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLEA*02: 01-abTCR-3 WMGRIDPGDSYTTYNPSFQGHVTISIDKSTNTAYLHWNSLKASDTA alphaMYYCARYYVSLVDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 24 anti-AFP158/HLA-QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPK A*02:01-abTCR-3LMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYT betaTGSRAVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 25 anti-AFP158/HLA-QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPK A*02:01-abTCR-4LMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYT alphaTGSRAVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 26 anti-AFP158/HLA-EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLE A*02:01-abTCR-4WMGRIDPGDSYTTYNPSFQGHVTISIDKSTNTAYLHWNSLKASDTA betaMYYCARYYVSLVDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 27 anti-AFP158/HLA-QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPK A*02: 01-abTCR-LMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYT 4MD alphaTGSRAVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 28 anti-AFP158/HLA-EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLE A*02:01-abTCR-WMGRIDPGDSYTTYNPSFQGHVTISIDKSTNTAYLHWNSLKASDTA 4MD betaMYYCARYYVSLVDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 29 anti-AFP158/HLA-QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPK A*02:01-abTCR-5LMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYT deltaTGSRAVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 30 anti-AFP158/HLA-EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLE A*02:01-abTCR-5WMGRIDPGDSYTTYNPSFQGHVTISIDKSTNTAYLHWNSLKASDTA gammaMYYCARYYVSLVDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEK S 31 anti-AFP158/HLA-QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPK A*02:01-abTCR-LMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYT 5MD deltaTGSRAVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTA KLFFL 32 anti-AFP158/HLA-EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLE A*02:01-abTCR-WMGRIDPGDSYTTYNPSFQGHVTISIDKSTNTAYLHWNSLKASDTA 5MD gammaMYYCARYYVSLVDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTA FCCNGEKS 33anti-AFP158/HLA- EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLEA*02:01-abTCR-6 WMGRIDPGDSYTTYNPSFQGHVTISIDKSTNTAYLHWNSLKASDTA deltaMYYCARYYVSLVDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTA KLFFL 34 anti-AFP158/HLA-QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPK A*02:01-abTCR-6LMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYT gammaTGSRAVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 35 anti-AFP158/HLA-EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLE A*02:01-abTCR-WMGRIDPGDSYTTYNPSFQGHVTISIDKSTNTAYLHWNSLKASDTA 6MD deltaMYYCARYYVSLVDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAV NFLLTAKLFFL 36anti-AFP158/HLA- QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKA*02:01-abTCR- LMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYT 6MD gammaTGSRAVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNG EKS 37 anti-AFP158/HLA-QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPK A*02:01-scFv CARLMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTGSRAVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAEVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLEWMGRIDPGDSYTTYNPSFQGHVTISIDKSTNTAYLHWNSLKASDTAMYYCARYYVSLVDIWGQGTLVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR 38IgVH domain of anti- EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLEAFP158/HLA- WMGRIDPGDSYTTYNPSFQGHVTISIDKSTNTAYLHWNSLKASDTAA*02:01 antibody MYYCARYYVSLVDIWGQGTLVTVSS 39 IgG1 CH1 domainASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSC 40IgVL domain of QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKAFP158/HLA- LMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTA*02:01 antibody TGSRAVFGGGTKLTVL 41 IgCL domainGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE GSTVEKTVAPTECS 42anti-CD19-abTCR- EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLE6MD delta WMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQVWGWQGGMYPRSNWWYNMDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLT VLGLRMLFAKTVAVNFLLTAKLFFL43 anti-CD19-abTCR- LPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLV6MD gamma VYDDSNRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSEYVVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGE KS 44 anti-CD19-scFv CARLPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSNRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSEYVVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQVWGWQGGMYPRSNWWYNMDSWGQGTLVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR45 IgVH domain of anti- EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLECD19 antibody WMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQVWGWQGGMYPRSNWWYNMDSWGQGTLVTVSS 46 IgVL domain of anti-LPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLV CD19 antibodyVYDDSNRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSS SEYVVFGGGTKLTVL 47fragment of CD28 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRS 48fragment of CD3-zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR 49Signal peptide METDTLLLWVLLLWVPGSTG 50 HA tag YPYDVPDYA 51 3x Flag tagDYKDHDGDYKDHDIDYKDDDDK 52 myc tag EQKLISEEDL 53 AFP158 FMNKFIYEI 54anti-CD19 clone 5 LPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVabTCR-6MD gamma VYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDYVVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGE KS 55anti-CD19 clone 5-9 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEabTCR-6MD delta WMGIIYPGDSDTRYSPFFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQVWGWQGGMYPRSNWWYNMDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLT VLGLRMLFAKTVAVNFLLTAKLFFL56 anti-CD19 clone 5-13 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEabTCR-6MD delta WMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQVWGWQGGMYPRSNWWYNLDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLT VLGLRMLFAKTVAVNFLLTAKLFFL57 IgVL domain of anti- LPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVCD19 antibody clone VYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSS 5SDYVVFGGGTKLTVL 58 IgVH domain of anti-EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLE CD19 antibody cloneWMGIIYPGDSDTRYSPFFQGQVTISADKSISTAYLQWSSLKASDTAM 5-9YYCARQVWGWQGGMYPRSNWWYNMDSWGQGTLVTVSS 59 IgVH domain of anti-EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLE CD19 antibody cloneWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAM 5-13YYCARQVWGWQGGMYPRSNWWYNLDSWGQGTLVTVSS 60 IgG2-0C CH1ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKV DKTVERK 61 IgG2-1C CH1ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKV DKTVERKC 62 IgG2-2C CH1ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKV DKTVERKCC 63 IgG3 CH1ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKV DKRVELKTP 64 IgG4 CH1ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKV DKRVESKYG 65 IgA1 CH1ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPS 66 IgA2 CH1ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPS QDVTVPCPVPPPPP 67IgD CH1 APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGE EKKKEKEKEEQEERETKTP 68IgE CH1 ASTQSPSVFPLTRCCKNIPSNATSVTLGCLATGYFPEPVMVTWDTGSLNGTTMTLPATTLTLSGHYATISLLTVSGAWAKQMFTCRVAHTPSS TDWVDNKTFS 69 IgM CH1GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPN GNKEKNVPLP 70CD28 co-stimulatory RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS fragment71 4-1BB co-stimulatory KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELfragment 72 IgVH domain of anti-QVQLVQSGAEVKKPGSSVKVSCKASGDTFSSYSISWVRQAPGQGLE NY-ESO-1/HLA-WMGRIIPILGIANYAQKYQGRVTLSADKSTSTSYMELNSLRSEDTAV A*02:01 antibodyYYCARDWSYSIDYWGQGTLVTVSS clone 35 73 IgVL domain of anti-QSVVTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKL NY-ESO-1/HLA-LIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDS A*02:01 antibodySLSAWVFGGGTKLTVLG clone 35 74 scFv linker SRGGGGSGGGGSGGGGSLEMA 75anti-CD19-cTCR delta EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQVWGWQGGMYPRSNWWYNLDSWGQGTLVTVSSRSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNF LLTAKLFFL 76anti-CD19-cTCR LPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLV gammaVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDYVVFGGGTKLTVLGDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEK S 77 TCRα constantPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITD domainKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 78 TCRβ constantEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWV domainNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 79 TCRδ constantSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIV domainISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTV AVNFLLTAKLFFL 80TCRγ constant DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQE domainKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 81 anti-AFP158/HLA-EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLE A*02:01 abTCR-7WMGRIDPGDSYTTYNPSFQGHVTISIDKSTNTAYLHWNSLKASDTA deltaMYYCARYYVSLVDIWGQGTLVTVSSRSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 82 anti-AFP158/HLA-QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPK A*02:01 abTCR-7LMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYT gammaTGSRAVFGGGTKLTVLDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEK S

What is claimed is:
 1. An antibody-T cell receptor (TCR) chimericmolecule (abTCR) that specifically binds to a target antigen,comprising: a) a first polypeptide chain comprising a firstantigen-binding domain comprising a V_(H) antibody domain and a first Tcell receptor domain (TCRD) comprising a first transmembrane domain of afirst TCR subunit; and b) a second polypeptide chain comprising a secondantigen-binding domain comprising a V_(L) antibody domain and a secondTCRD comprising a second transmembrane domain of a second TCR subunit,wherein the V_(H) domain of the first antigen-binding domain and theV_(L) domain of the second antigen-binding domain form anantigen-binding module that specifically binds to the target antigen,wherein the first TCRD and the second TCRD form a T cell receptor module(TCRM) that is capable of recruiting at least one TCR-associatedsignaling module, wherein the target antigen is a complex comprising apeptide and an MHC protein, and wherein: (i) the first TCR subunit is aTCR α chain, and the second TCR subunit is a TCR β chain; (ii) the firstTCR subunit is a TCR β chain, and the second TCR subunit is a TCR αchain; (iii) the first TCR subunit is a TCR γ chain, and the second TCRsubunit is a TCR δ chain; or (iv) the first TCR subunit is a TCR δchain, and the second TCR subunit is a TCR γ chain.
 2. The abTCR ofclaim 1, wherein the first polypeptide chain further comprises a firstpeptide linker between the first antigen-binding domain and the firstTCRD, and the second polypeptide chain further comprises a secondpeptide linker between the second antigen-binding domain and the secondTCRD.
 3. The abTCR of claim 2, wherein the first and/or second peptidelinkers comprise, individually, a constant domain or fragment thereoffrom an immunoglobulin or T cell receptor subunit.
 4. The abTCR of claim3, wherein the first and/or second peptide linkers comprise,individually, a CH1, CH2, CH3, CH4 or CL antibody domain, or a fragmentthereof.
 5. The abTCR of claim 3, wherein the first and/or secondpeptide linkers comprise, individually, a Cα, Cβ, Cγ, or Cδ TCR domain,or a fragment thereof.
 6. The abTCR of claim 1, wherein the first TCRDfurther comprises a first connecting peptide or fragment thereof of aTCR subunit N-terminal to the first transmembrane domain; and/or whereinthe second TCRD further comprises a second connecting peptide orfragment thereof of a TCR subunit N-terminal to the second transmembranedomain.
 7. The abTCR of claim 1, wherein the first TCRD furthercomprises a first TCR intracellular domain comprising a TCRintracellular sequence C-terminal to the first transmembrane domain;and/or wherein the second TCRD further comprises a second TCRintracellular domain comprising a TCR intracellular sequence C-terminalto the second transmembrane domain.
 8. The abTCR of claim 1, wherein thefirst polypeptide chain further comprises a first accessoryintracellular domain comprising a co-stimulatory intracellular signalingsequence C-terminal to the first transmembrane domain; and/or whereinthe second polypeptide chain further comprises a second accessoryintracellular domain comprising a co-stimulatory intracellular signalingsequence C-terminal to the second transmembrane domain.
 9. The abTCR ofclaim 1, wherein the peptide in the target antigen complex is derivedfrom a protein selected from the group consisting of WT-1, AFP,HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, and PSA.
 10. The abTCR ofclaim 1, wherein the TCR-associated signaling module is selected fromthe group consisting of CD3δε, CD3γε, and ζζ.
 11. Nucleic acid(s)encoding the first and second polypeptide chains of the abTCR ofclaim
 1. 12. An effector cell presenting on its surface the abTCR ofclaim
 1. 13. The effector cell of claim 12, wherein the effector celldoes not express the first TCR subunit and/or the second TCR subunit.14. The effector cell of claim 12, wherein effector cell is modified toblock or decrease the expression of a first endogenous TCR subunitand/or a second endogenous TCR subunit.
 15. A method of killing a targetcell presenting a target antigen, comprising contacting the target cellwith the effector cell of claim 12, wherein the abTCR specifically bindsto the target antigen.
 16. A pharmaceutical composition comprising theeffector cell of claim 12, and a pharmaceutically acceptable carrier.17. A method of treating a target antigen-associated disease in anindividual in need thereof comprising administering to the individual aneffective amount of the pharmaceutical composition of claim
 16. 18. Themethod of claim 17, wherein the target antigen-associated disease is acancer.
 19. The method of claim 18, wherein the cancer comprises a solidtumor.
 20. The method of claim 19, wherein the cancer is selected fromthe group consisting of adrenocortical carcinoma, bladder cancer, breastcancer, cervical cancer, cholangiocarcinoma, colorectal cancers,esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, headand neck cancer, kidney cancer, lung cancer, melanoma, mesothelioma,pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma,ovarian cancer, prostate cancer, sarcoma, stomach cancer, uterine cancerand thyroid cancer.
 21. The method of claim 17, wherein the targetantigen-associated disease is a viral infection.
 22. The method of claim21, wherein the viral infection is caused by a virus selected from thegroup consisting of Cytomegalovirus (CMV), Epstein-Barr Virus (EBV),Hepatitis B Virus (HBV), Kaposi's Sarcoma associated herpesvirus (KSHV),Human papillomavirus (HPV), Molluscum contagiosum virus (MCV), Human Tcell leukemia virus 1 (HTLV-1), HIV (Human immunodeficiency virus), andHepatitis C Virus (HCV).